U.S. patent application number 16/919919 was filed with the patent office on 2021-01-28 for conjugates containing hydrophilic spacer linkers.
The applicant listed for this patent is Endocyte, Inc.. Invention is credited to Paul Joseph Kleindl, Christopher Paul Leamon, Hari Krishna R. Santhapuram, Iontcho Radoslavov Vlahov, Kevin Yu Wang, Fei You.
Application Number | 20210024581 16/919919 |
Document ID | / |
Family ID | 1000005134599 |
Filed Date | 2021-01-28 |
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United States Patent
Application |
20210024581 |
Kind Code |
A1 |
Leamon; Christopher Paul ;
et al. |
January 28, 2021 |
CONJUGATES CONTAINING HYDROPHILIC SPACER LINKERS
Abstract
Described herein are compositions and methods for use in
targeted drug delivery using cell receptor binding drug delivery
conjugates containing hydrophilic spacer linkers for use in
imaging, diagnosing, and/or treating diseases and disease states
caused by pathogenic cell populations.
Inventors: |
Leamon; Christopher Paul;
(West Lafayette, IN) ; Vlahov; Iontcho Radoslavov;
(West Lafayette, IN) ; Santhapuram; Hari Krishna R.;
(West Lafayette, IN) ; Kleindl; Paul Joseph;
(Lebanon, IN) ; Wang; Kevin Yu; (Fishers, IN)
; You; Fei; (West Lafayette, IN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Endocyte, Inc. |
West Lafayette |
IN |
US |
|
|
Family ID: |
1000005134599 |
Appl. No.: |
16/919919 |
Filed: |
July 2, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15431157 |
Feb 13, 2017 |
10738086 |
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16919919 |
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14820777 |
Aug 7, 2015 |
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15431157 |
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12666712 |
Dec 24, 2009 |
9138484 |
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PCT/US08/68093 |
Jun 25, 2008 |
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14820777 |
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61036186 |
Mar 13, 2008 |
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60946092 |
Jun 25, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/545 20170801;
C07K 9/003 20130101; C07H 15/24 20130101; A61K 47/65 20170801; A61K
47/60 20170801 |
International
Class: |
C07K 9/00 20060101
C07K009/00; A61K 47/54 20060101 A61K047/54; A61K 47/65 20060101
A61K047/65; A61K 47/60 20060101 A61K047/60; C07H 15/24 20060101
C07H015/24 |
Claims
1.-39. (canceled)
40. A compound of the formula B-L-A wherein B is of the formula
##STR00218## wherein ** represents a covalent bond to L; L is a
releasable linker comprising a first hydrophilic spacer linker
portion is of the formula ##STR00219## wherein R is H, alkyl,
cycloalkyl, or arylalkyl; m is an independently selected integer
from 1 to 3; n is an integer from 1 to 6, p is an integer from 1 to
5, ** represents a covalent bond to B; and * represents a covalent
bond a first spacer linker portion of the formula ##STR00220##
wherein * represents a covalent bond to the first hydrophilic
spacer linker portion, and *** represents a covalent bond to a
second hydrophilic spacer linker portion of the formula
##STR00221## wherein R is H, alkyl, cycloalkyl, or arylalkyl; m is
an integer from 1 to 3; n is an integer from 1 to 6, p is an
integer from 1 to 5, *** represents a covalent bond to the first
spacer linker portion, and * represents a covalent bond to a second
spacer linker portion of the formula ##STR00222## wherein *
represents a covalent bond to the second hydrophilic spacer linker
portion, and **** represents a covalent bond to an additional
hydrophilic spacer linker portion or an additional spacer linker
portion; and a releasable disulfide portion of the formula
##STR00223## wherein ** represents a covalent bond to in the
additional hydrophilic spacer linker portion or the additional
spacer linker portion, and * represents a covalent bond to the rest
of the compound; and A is therapeutic agent selected from the group
consisting of DAVLBH, bortezomib, thiobortezomib, a tubulysin,
aminopterin, rapamycin, paclitaxel, docetaxel, doxorubicin,
daunorubicin, everolimus, .alpha.-amanatin, verucarin, didemnin B,
geldanomycin, purvalanol A, everolimus, ispinesib, budesonide, and
dasatinib.
41. The compound of claim 40, wherein p in the first hydrophilic
spacer linker portion is 1.
42. The compound of claim 41, wherein p in the second hydrophilic
spacer linker portion is 1.
43. The compound of claim 42, wherein m in the first hydrophilic
spacer linker portion is 2.
44. The compound of claim 43, wherein m in the second hydrophilic
spacer linker portion is 2.
45. The compound of claim 44, wherein n in the first hydrophilic
spacer linker portion, when present, is 5.
46. The compound of claim 45, wherein n in the second hydrophilic
spacer linker portion, when present, is 5.
47. The compound of claim 46, wherein R in the first hydrophilic
spacer linker portion, when present, is H.
48. The compound of claim 47, wherein R in the second hydrophilic
spacer linker portion, when present, is H.
49. The compound of claim 48, wherein A is DAVLBH.
50. The compound of claim 48, wherein A is a tubulysin.
51. A compound of the formula B-L-A wherein B is of the formula
##STR00224## wherein ** represents a covalent bond to L; L is a
releasable linker comprising a first hydrophilic spacer linker
portion is of the formula ##STR00225## wherein R is H; m is 2; n is
5, p is 1, ** represents a covalent bond to B; and * represents a
covalent bond a first spacer linker portion of the formula
##STR00226## wherein * represents a covalent bond to the first
hydrophilic spacer linker portion, and *** represents a covalent
bond to a second hydrophilic spacer linker portion of the formula
##STR00227## wherein R is H; m is 2; n is 5, p is 1, *** represents
a covalent bond to the first spacer linker portion, and *
represents a covalent bond to a second spacer linker portion of the
formula ##STR00228## wherein * represents a covalent bond to the
second hydrophilic spacer linker portion, and **** represents a
covalent bond to an additional hydrophilic spacer linker portion or
an additional spacer linker portion; and a releasable-disulfide
portion of the formula ##STR00229## wherein ** represents a
covalent bond to in the additional hydrophilic spacer linker
portion or the additional spacer linker portion, and * represents a
covalent bond to the rest of the compound; and A is an
anti-inflammatory agent.
52. A pharmaceutical composition comprising (a) an effective amount
of a compound of claim 40, and optionally one or more of a carrier,
a diluent, or an excipient.
53. A pharmaceutical composition comprising (a) an effective amount
of a compound of claim 48, and optionally one or more of a carrier,
a diluent, or an excipient.
54. A pharmaceutical composition comprising (a) an effective amount
of a compound of claim 51, and optionally one or more of a carrier,
a diluent, or an excipient.
55. A method for treating a disease in a patient comprising
administering an effective amount of the compound of claim 40 to
the patient.
56. The method of claim 55, wherein the disease is
inflammation.
57. A method for treating a disease in a patient comprising
administering an effective amount of the compound of claim 48 to
the patient.
58. The method of claim 57, wherein the disease is
inflammation.
59. A method for treating inflammation in a patient comprising
administering an effective amount of the compound of claim 51 to
the patient.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority under 35
U.S.C. .sctn. 119(e) of U.S. provisional patent application Ser.
Nos. 60/946,092 and 61/036,186, filed Jun. 25, 2007 and Mar. 13,
2008, respectively; the disclosures of which are incorporated
herein in their entirety by reference.
TECHNICAL FIELD
[0002] The present invention relates to compositions and methods
for use in targeted drug delivery. More particularly, the invention
is directed to cell-surface receptor binding drug delivery
conjugates containing hydrophilic spacer linkers for use in
treating disease states caused by pathogenic cell populations and
to methods and pharmaceutical compositions that use and include
such conjugates.
BACKGROUND
[0003] The mammalian immune system provides a means for the
recognition and elimination of tumor cells, other pathogenic cells,
and invading foreign pathogens. While the immune system normally
provides a strong line of defense, there are many instances where
cancer cells, other pathogenic cells, or infectious agents evade a
host immune response and proliferate or persist with concomitant
host pathogenicity. Chenotherapeutic agents and radiation therapies
have been developed to eliminate, for example, replicating
neoplasms. However, many of the currently available
chemotherapeutic agents and radiation therapy regimens have adverse
side effects because they work not only to destroy pathogenic
cells, but they also affect normal host cells, such as cells of the
hematopoietic system. The adverse side effects of these anticancer
drugs highlight the need for the development of new therapies
selective for pathogenic cell populations and with reduced host
toxicity.
[0004] Researchers have developed therapeutic protocols for
destroying pathogenic cells by targeting cytotoxic compounds to
such cells. Many of these protocols utilize toxins conjugated to
antibodies that bind to antigens unique to or overexpressed by the
pathogenic cells in an attempt to minimize delivery of the toxin to
normal cells. Using this approach, certain immunotoxins have been
developed consisting of antibodies directed to specific antigens on
pathogenic cells, the antibodies being linked to toxins such as
ricin, Pseudomonas exotoxin, Diptheria toxin, and tumor necrosis
factor. These immunotoxins target pathogenic cells, such as tumor
cells, bearing the specific antigens recognized by the antibody
(Olsnes, S., Immunol. Today, 10, pp. 291-295, 1989; Melby, E. L.,
Cancer Res., 53(8), pp. 1755-1760, 1993; Better, M. D., PCT
Publication Number WO 91/07418, published May 30, 1991).
[0005] Another approach for targeting populations of pathogenic
cells, such as cancer cells or foreign pathogens, in a host is to
enhance the host immune response against the pathogenic cells to
avoid the need for administration of compounds that may also
exhibit independent host toxicity. One reported strategy for
immunotherapy is to bind antibodies, for example, genetically
engineered multimeric antibodies, to the surface of tumor cells to
display the constant region of the antibodies on the cell surface
and thereby induce tumor cell killing by various immune-system
mediated processes (De Vita, V. T., Biologic Therapy of Cancer, 2d
ed. Philadelphia, Lippincott, 1995; Soulillou, J. P., U.S. Pat. No.
5,672,486). However, these approaches have been complicated by the
difficulties in defining tumor-specific antigens. Accordingly,
additional compounds and methods are needed for selectively
targeting pathogenic cell populations.
SUMMARY OF THE INVENTION
[0006] It has been discovered that therapeutic agents, diagnostic
agents, and imaging agents may be conjugated to other compounds to
control or alter their behavior, biodistribution, metabolism,
and/or clearance in vivo. In one illustrative embodiment of the
invention, conjugates of compounds are described that include a
hydrophilic spacer linker. In one aspect, conjugates of compounds
are described that include both a hydrophilic spacer linker and a
targeting ligand. Illustrative of such conjugates are compounds of
the following formula described herein
B-L-A
wherein B is a receptor binding ligand that binds to a target cell
receptor, L is a linker that comprises one or more hydrophilic
spacer linkers, and A is a diagnostic, therapeutic, or imaging
agent that is desirably delivered to the cell.
[0007] In another embodiment, non-receptor binding targeted
compounds of the following formula are described herein:
L-A
where L is a linker that comprises one or more hydrophilic spacer
linkers and A is diagnostic, therapeutic, or imaging agent. In one
variation, the linker L does not include a releasable linker. In
another variation, the linker L includes a releasable linker. In
another embodiment, at least one of the hydrophilic spacer linkers
is formed from or includes at least one carbohydrate. In one
variation, the carbohydrate forms part of the linker chain
connecting B and A. In another variation, the carbohydrate forms
part of a side chain attached to the linker chain connecting B and
A.
[0008] It is appreciated that in each of the above embodiments,
more than one receptor binding ligand B may be attached to the
linkers described herein. It is further appreciated that more than
one agent A may be attached to the linkers described herein. Such
multi-ligand and/or multi-drug conjugates are also described
herein, where the linker comprises a hydrophilic spacer linker.
[0009] In another embodiment, compounds are described herein that
have reduced uptake by the liver and are less likely to be cleared
by the liver. In one aspect, such compounds are preferentially
cleared by the renal processes as compared to hepatic
processes.
[0010] The agent or agents A include therapeutic drugs, diagnostic
agents, imaging agents, and any other compound that is desirably or
advantageously delivered to a cell by targeting a cell receptor.
Illustrative drugs include cytotoxic drugs, anti-inflammatory
agents, and the like. Illustrative diagnostic agents and imaging
agents include PET imaging agents, fluorescent imaging agents,
radioligands, radioligand complexing agents, and others.
[0011] In the embodiments of compounds, compositions, and methods
described herein, the cells that may be targeted with the
therapeutic, diagnostic, and/or imaging agents A include a wide
variety, such as but not limited to cancer cells, bacterial cells,
tumor cells, monocytes, activated macrophages, progenitor cells,
such as endothelial progenitor cells, other inflammatory cells,
atherosclerotic plaques, infections, and others. The targeting of
the cell is accomplished by the appropriate selection of a cell
receptor binding ligand B. It is appreciated that selective or
specific targeting of a cell in vivo may be accomplished by
selecting a receptor that is preferentially expressed or
overexpressed by the target cell. Illustratively, the target cell
preferentially expresses or overexpresses a vitamin receptor, such
as folate receptors.
[0012] In another embodiment, the conjugates described herein are
included in pharmaceutical compositions in amounts effective to
treat diseases and disease states associated with pathogenic
populations of cells.
[0013] In another embodiment, the conjugates described herein, and
pharmaceutical compositions containing them are used in methods for
treating diseases and disease states associated with pathogenic
populations of cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 shows the relative binding affinity of EC234, DPM for
folic acid (.circle-solid.) and EC0234 (.box-solid.).
[0015] FIG. 2 shows the activity of EC0258 against KB cells (2 h
pulse/72 h chase) for EC258 (.circle-solid.) and EC258+excess folic
acid (.largecircle.).
[0016] FIG. 3A shows the effect of EC0234 and EC0246 against M109
tumors in mice, untreated controls (.box-solid.), EC145 standard
(TTW 3 .mu.mol/kg, 3 wks) (.circle-solid.), EC0234 (TIW 3
.mu.mol/kg, 3 wks) (), and EC0246 (TIW 3 mol/kg, 3 wks)
(.tangle-solidup.).
[0017] FIG. 3B shows the effect of EC0234 and EC0246 on percentage
body weight change, untreated controls (.box-solid.), EC145
standard (TIW 3 .mu.mol/kg, 3 wks) (.circle-solid.), EC0234 (TIW 3
.mu.mol/kg, 3 wks) (), and EC0246 (TIW 3 mol/kg, 3 wks)
(.tangle-solidup.); indicating that no gross toxicity was observed
during treatment.
[0018] FIG. 4A shows the effect on KB tumor volume in mice of
EC0396 (), EC145 (.tangle-solidup.) and PBS control (.box-solid.)
dosed at 2 mol/kg TIW for two weeks (the vertical line indicates
the last dosing day).
[0019] FIG. 4B shows the effect on percentage body weight change of
EC0396 (), EC145 (.tangle-solidup.) and PBS control (.box-solid.)
dosed at 2 mol/kg TTW for two weeks (the vertical line indicates
the last dosing day); indicating that no gross toxicity was
observed during treatment.
[0020] FIG. 5A shows the effect on KB tumor volume of EC0400
(.circle-solid.), EC145 (.tangle-solidup.) and PBS control
(.box-solid.) dosed at 2 mol/kg TIW for two weeks (the vertical
line indicates the last dosing day).
[0021] FIG. 5B shows the effect on percentage body weight change of
EC0400 (.circle-solid.), EC145 (.tangle-solidup.) and PBS control
(.box-solid.) dosed at 2 .mu.mol/kg TIW for two weeks (the vertical
line indicates the last dosing day); indicating that no gross
toxicity was observed during treatment.
[0022] FIG. 6A shows the effect on tumor volume of EC0429
(.gradient.) and EC145 (.tangle-solidup.), dosed at 2 .mu.mol/kg
TIW for two weeks (the vertical line indicates the last dosing day)
compared to untreated controls (.circle-solid.) for M109 tumors in
Balb/c mice.
[0023] FIG. 6B shows the effect on percentage body weight change
EC0429 (.gradient.) and EC145 (.tangle-solidup.), dosed at 2 mol/kg
TIW for two weeks (the vertical line indicates the last dosing day)
compared to untreated controls (.circle-solid.); indicating that no
gross toxicity was observed during treatment.
[0024] FIG. 7A shows the effect on tumor volume of EC0434
(.gradient.) and EC145 (.tangle-solidup.), dosed at 2 .mu.mol/kg
TIW for two weeks (the vertical line indicates the last dosing day)
compared to untreated controls (.circle-solid.) for s.c. M109
tumors in Balb/c mice.
[0025] FIG. 7B shows the effect on percentage body weight change of
EC0434 (.gradient.) and EC145 (.tangle-solidup.), dosed at 2
.mu.mol/kg TIW for two weeks (the vertical line indicates the last
dosing day) compared to untreated controls (.circle-solid.);
indicating that no gross toxicity was observed during
treatment.
[0026] FIG. 8A shows the effect on tumor volume of EC0305
(.circle-solid.), EC0436 () and PBS control (.box-solid.) dosed at
2 .mu.mol/kg TIW for two weeks (the vertical line indicates the
last dosing day) for s.c. M109 tumors in Balb/c mice.
[0027] FIG. 8B shows the effect on percentage body weight change of
EC0305 (.circle-solid.), EC0436 () and PBS control (.circle-solid.)
dosed at 2 mol/kg TIW for two weeks (the vertical line indicates
the last dosing day); indicating that no gross toxicity was
observed during treatment.
[0028] FIG. 9 shows the percentage body weight change of Balb/c
mice having s.c. M109 tumors treated intravenously three times in a
week for one week with PBS (untreated controls) (.circle-solid.)
EC0436 (TIW 2 .mu.mol/kg) (.tangle-solidup.), EC0436 (TIW 2.5
.mu.mol/kg) (), EC0436 (TIW 3 .mu.mol/kg) (.circle-solid.), EC0305
(TIW 2 mol/kg) (.DELTA.), EC0305 (TIW 2.5 .mu.mol/kg) (.gradient.),
and EC0305 (TIW 3 mol/kg) (.quadrature.).
[0029] FIG. 10A shows the effect on s.c. KB tumors in nu/nu mice by
EC0565 at 3 mol/kg (qdx5 for two weeks) (.circle-solid.), compared
to PBS treated controls (.box-solid.). From the data, a Log Cell
Kill (LCK) value of 1.2 can be determined (values greater than
about 0.7 are indicative of an active anti-cancer compound).
[0030] FIG. 10B shows the effect on on percentage body weight
change by EC0565 at 3 .mu.mol/kg (qdx5 for two weeks)
(.circle-solid.), compared to PBS treated controls (.box-solid.);
indicating that no gross toxicity was observed during
treatment.
[0031] FIG. 11 shows the total DAVLBH biliary excretion from
various DAVLBH conjugates at 2 .mu.mol/kg i.v. bolus in a
hepatobiliary excretion in bile duct assay in cannulated rats. The
percent of total dose in the bile was measured for EC145=8.7%
(.circle-solid.), EC0409=7.9% (.diamond-solid.), EC0429=8.6%
(.box-solid.), EC0434=2.8% (). In addition, EC145 shows an AUC=1092
(.circle-solid.); last time point collected was 139 min; and EC0434
shows an AUC=260 (.box-solid.); the 120, 135, and 360 minute time
points were all below level of quantitation, i.e. <0.65 M.
[0032] FIG. 12 shows the effect of ribose-based spacers on bile
clearance and the impact of extended derivatization. The numbers
above bars correspond to the number of hydrophilic spacers in the
linker.
[0033] FIG. 13 shows that EC0565 induces dose-responsive inhibition
of RPS6 and p70S6K in KB cells (1 h pulse/4 h chase) using a 30 min
camera exposure, where C=Control (untreated cells); FAC=Folic acid
control (100 .mu.M).
[0034] FIG. 14 shows the cytotoxicity of bortezomib versus the
methylthiol bortezomib derivative (EC0501). IC.sub.50 bortezomib,
20 nM (.circle-solid.); EC0501, 240 nM (.box-solid.).
[0035] FIG. 15 shows that hydrophilic spacer linkers enable
specific activity of mono- and bis-thio-velcade folate conjugates
against RAW264.7 cells. Cell viability after a 5 h pulse, followed
by a 72 h chase (MTT); bortezomib (.box-solid.), EC0501
(.quadrature.), EC0522 (.tangle-solidup.), EC0522 plus excess folic
acid (.gradient.).
[0036] FIG. 16 shows cell viability (5 h pulse/72 h chase) (XTT)
after treatment with EC0595 (13 nM IC50) (), EC0595 plus excess
folic acid (.gradient.), bortezomib (.box-solid.), EC0525 (46 nM
IC50) (.circle-solid.), EC0525 plus excess folic acid
(.largecircle.).
[0037] FIG. 17 shows cell viability after a 24 h incubation (XTT)
with bortezomib (.box-solid.), EC0587 (.circle-solid.), EC0587 plus
excess folic acid (o).
[0038] FIG. 18 shows inhibition of LPS stimulated proteosome
activity in RAW 264.7 cells (5 h pulse/24 h chase), LPS 100 ng/mL,
30 m 20S proteosome/substrate reaction time by bortezomib
(.box-solid.), EC0522 (), EC0522 plus excess folic acid
(.gradient.), EC0525 (.circle-solid.), EC0525 plus excess folic
acid (.largecircle.), EC0595 (.diamond-solid.), EC0595 plus excess
folic acid (.quadrature.); IC.sub.50 is ca. 30 nM for EC0595 and
EC0525.
[0039] FIG. 19 shows activity against RAW cells (5 h pulse/72 h
chase) after treatment with .alpha.-amantin (.box-solid.), EC0592
(IC.sub.50 3.7 nM) (.circle-solid.), EC0592 plus excess folic acid
(.largecircle.).
DETAILED DESCRIPTION
[0040] Drug delivery conjugates are described herein consisting of
a receptor binding ligand (B), a polyvalent linker (L) comprising
one or more hydrophilic spacer linkers, and a diagnostic,
therapeutic, or imaging agent (A) that is desirably delivered to a
cell. The binding ligand (B) is covalently attached to the
polyvalent linker (L), and the diagnostic, therapeutic, or imaging
agent (A), or analog or derivative thereof, is also covalently
attached to the polyvalent linker (L). It is to be understood that
the diagnostic, therapeutic, or imaging agent (A) includes analogs
and derivatives thereof that are attached to the linker (L). The
polyvalent linker (L) comprises one or more spacer linkers and/or
releasable linkers, and combinations thereof, in any order. In one
variation, releasable linkers, and optional spacer linkers are
covalently bonded to each other to form the linker. In another
variation, a releasable linker is directly attached to the agent
(A), or analog or derivative thereof. In another variation, a
releasable linker is directly attached to the binding ligand. In
another variation, either or both the binding ligand and the agent
(A), or analog or derivative thereof, is attached to a releasable
linker through one or more spacer linkers. In another variation,
each of the binding ligand and the agent (A), or analog or
derivative thereof, is attached to a releasable linker, each of
which may be directly attached to each other, or covalently
attached through one or more spacer linkers.
[0041] From the foregoing, it should be appreciated that the
arrangement of the binding ligand, and the agent (A), or analog or
derivative thereof, and the various releasable and optional spacer
linkers may be varied widely. In one aspect, the binding ligand,
and the agent (A), or analog or derivative thereof, and the various
releasable and optional spacer linkers are attached to each other
through heteroatoms, such as nitrogen, oxygen, sulfur, phosphorus,
silicon, and the like. In variations, the heteroatoms, excluding
oxygen, may be in various states of oxidation, such as N(OH), S(O),
S(O).sub.2, P(O), P(O).sub.2, P(O).sub.3, and the like. In other
variation, the heteroatoms may be grouped to form divalent
radicals, such as for example hydroxylamines, hydrazines,
hydrazones, sulfonates, phosphinates, phosphonates, and the like,
including radicals of the formulae --(NHR.sup.1NHR.sup.2)--,
--SO--, --(SO.sub.2)--, and --N(R.sup.3)O--, wherein R.sup.1,
R.sup.2, and R.sup.3 are each independently selected from hydrogen,
alkyl, aryl, arylalkyl, substituted aryl, substituted arylalkyl,
heteroaryl, substituted heteroaryl, and alkoxyalkyl. In another
variation, more than one binding ligand is attached to the
polyvalent linker. In another variation, more than one agent (A) is
attached to the polyvalent linker. In another variation, more than
one binding ligand and more than one agent (A) is attached to the
polyvalent linker.
[0042] In one embodiment, the receptor binding ligand is a vitamin
receptor binding ligand such as a vitamin, or an analog or a
derivative thereof, capable of binding to vitamin receptors. In
another embodiment, the binding ligand is a vitamin, or analog or
derivative thereof, attached to a releasable linker which is
attached to the drug through a linker that is formed from one or
more spacer linkers and/or releasable linkers and/or hydrophilic
spacer linkers. In one variation, both the drug and the vitamin, or
analog or derivative thereof, can each be attached to spacer
linkers, where the spacer linkers are attached to each other
through one or more releasable linkers. In addition, both the drug
and the vitamin, or analog or derivative thereof, can each be
attached to one or more releasable linkers, where the releasable
linkers are attached to each other or through a spacer linker. Each
of these radicals may be connected through existing or additional
heteroatoms on the binding ligand, agent A, or releasable,
hydrophilic spacer, or additional spacer linker.
[0043] The binding site for the binding ligand (B) can include
receptors for any binding ligand (B), or a derivative or analog
thereof, capable of specifically binding to a receptor wherein the
receptor or other protein is uniquely expressed, overexpressed, or
preferentially expressed by a population of pathogenic cells. A
surface-presented protein uniquely expressed, overexpressd, or
preferentially expressed by the pathogenic cells is typically a
receptor that is either not present or present at lower
concentrations on non-pathogenic cells providing a means for
selective elimination, labeling or diagnosis of the pathogenic
cells. The binding ligand drug delivery conjugates may be capable
of high affinity binding to receptors on cancer cells or other
types of pathogenic cells. The high affinity binding can be
inherent to the binding ligand or the binding affinity can be
enhanced by the use of a chemically modified ligand (e.g., an
analog or a derivative of a vitamin).
[0044] The binding ligand drug delivery conjugates described herein
can be formed from, for example, a wide variety of vitamins or
receptor-binding vitamin analogs/derivatives, linkers, and drugs.
The binding ligand drug delivery conjugates described herein are
capable of selectively targeting a population of pathogenic cells
in the host animal due to preferential expression of a receptor for
the binding ligand, such as a vitamin, accessible for ligand
binding, on the pathogenic cells. Illustrative vitamin moieties
that can be used as the binding ligand (B) include carnitine,
inositol, lipoic acid, pyridoxal, ascorbic acid, niacin,
pantothenic acid, folic acid, riboflavin, thiamine, biotin, vitamin
B.sub.12, other water soluble vitamins, the B vitamins, and the
lipid soluble vitamins A, D, E and K. These vitamins, and their
receptor-binding analogs and derivatives, constitute an
illustrative targeting entity that can be coupled with the drug by
a bivalent linker (L) to form a binding ligand (B) drug delivery
conjugate as described herein. The term vitamin is understood to
include vitamin analogs and/or derivatives, unless otherwise
indicated. Illustratively, pteroic acid which is a derivative of
folate, biotin analogs such as biocytin, biotin sulfoxide,
oxybiotin and other biotin receptor-binding compounds, and the
like, are considered to be vitamins, vitamin analogs, and vitamin
derivatives. It should be appreciated that vitamin analogs or
derivatives as described herein refer to vitamins that incorporates
an heteroatom through which the vitamin analog or derivative is
covalently bound to the bivalent linker (L).
[0045] Illustrative vitamin moieties include folic acid, biotin,
riboflavin, thiamine, vitamin B.sub.12, and receptor-binding
analogs and derivatives of these vitamin molecules, and other
related vitamin receptor binding molecules.
[0046] In another embodiment, the cell receptor is a folate
receptor, and the targeting ligand B is a folate receptor binding
ligand. In another embodiment, B is a folate, such as folic acid,
or an analog or derivative of folic acid that binds to folic acid
receptors. It is to be understood as used herein, that the term
folate is used both individually and collectively to refer to folic
acid itself, and/or to such analogs and derivatives of folic acid
that are capable of binding to folate receptors. In another
embodiment, B is a compound capable of selectively or specifically
binding to a folate receptor, such as an antibody.
[0047] Illustrative embodiments of folate analogs and/or
derivatives include folinic acid, pteropolyglutamic acid, and
folate receptor-binding pteridines such as tetrahydropterins,
dihydrofolates, tetrahydrofolates, and their deaza and dideaza
analogs. The terms "deaza" and "dideaza" analogs refer to the
art-recognized analogs having a carbon atom substituted for one or
two nitrogen atoms in the naturally occurring folic acid structure,
or analog or derivative thereof. For example, the deaza analogs
include the 1-deaza, 3-deaza, 5-deaza, 8-deaza, and 10-deaza
analogs of folate. The dideaza analogs include, for example,
1,5-dideaza, 5,10-dideaza, 8,10-dideaza, and 5,8-dideaza analogs of
folate. Other folates useful as complex forming ligands include the
folate receptor-binding analogs aminopterin, amethopterin
(methotrexate), N.sup.10-methylfolate, 2-deamino-hydroxyfolate,
deaza analogs such as 1-deazamethopterin or 3-deazamethopterin, and
3',5'-dichloro-4-amino-4-deoxy-N.sup.10-methylpteroylglutamic acid
(dichloromethotrexate). The foregoing folic acid analogs and/or
derivatives are conventionally termed folates, reflecting their
ability to bind with folate-receptors, and such ligands when
conjugated with exogenous molecules are effective to enhance
transmembrane transport, such as via folate-mediated endocytosis as
described herein. Other suitable binding ligands capable of binding
to folate receptors to initiate receptor mediated endocytotic
transport of the complex include antibodies to the folate receptor.
An exogenous molecule in complex with an antibody to a folate
receptor is used to trigger transmembrane transport of the
complex.
[0048] Additional analogs of folic acid that bind to folic acid
receptors are described in US Patent Application Publication Serial
Nos. 2005/0227985 and 2004/0242582, the disclosures of which are
incorporated herein by reference. Illustratively, such folate
analogs have the general formula:
##STR00001##
wherein X and Y are each-independently selected from the group
consisting of halo, R.sup.2, OR.sup.2, SR.sup.3, and
NR.sup.4R.sup.5;
[0049] U, V, and W represent divalent moieties each independently
selected from the group consisting of --(R.sup.6a)C.dbd., --N.dbd.,
(R.sup.6a)C(R.sup.7a)--, and --N(R.sup.4a)--; Q is selected from
the group consisting of C and CH; T is selected from the group
consisting of S, O, N, and --C.dbd.C--;
[0050] A.sup.1 and A.sup.2 are each independently selected from the
group consisting of oxygen, sulfur, --C(Z)--, --C(Z)O--, --OC(Z)--,
--N(R.sup.4b)--, --C(Z)N(R.sup.4b)--, --N(R.sup.4b)C(Z)--,
--OC(Z)N(R.sup.4b)--, --N(R.sup.4b)C(Z)O--,
--N(R.sup.4b)C(Z)N(R.sup.5b)--, --S(O)--, --S(O).sub.2--,
--N(R.sup.4a)S(O)--, --C(R.sup.6b)(R.sup.7b)--, --N(C.ident.CH)--,
--N(CH.sub.2C.ident.CH)--, C.sub.1-C.sub.12 alkylene, and
C.sub.1-C.sub.12 alkyeneoxy, where Z is oxygen or sulfur;
[0051] R.sup.1 is selected from the group consisting of hydrogen,
halo, C.sub.1-C.sub.12 alkyl, and C.sub.1-C.sub.12 alkoxy; R.sup.2,
R.sup.3, R.sup.4, R.sup.4a, R.sup.4b, R.sup.5, R.sup.5b, R.sup.6b,
and R.sup.7b are each independently selected from the group
consisting of hydrogen, halo, C.sub.1-C.sub.12 alkyl,
C.sub.1-C.sub.12 alkoxy, C.sub.1-C.sub.12 alkanoyl,
C.sub.1-C.sub.12 alkenyl, C.sub.1-C.sub.12 alkynyl,
(C.sub.1-C.sub.12 alkoxy)carbonyl, and (C.sub.1-C.sub.12
alkylamino)carbonyl;
[0052] R.sup.6 and R.sup.7 are each independently selected from the
group consisting of hydrogen, halo, C.sub.1-C.sub.12 alkyl, and
C.sub.1-C.sub.12 alkoxy; or, R.sup.6 and R.sup.7 are taken together
to form a carbonyl group; R.sup.6a and R.sup.7a are each
independently selected from the group consisting of hydrogen, halo,
C.sub.1-C.sub.12 alkyl, and C.sub.1-C.sub.12 alkoxy; or R.sup.6a
and R.sup.7a are taken together to form a carbonyl group;
[0053] L is a divalent linker as described herein; and
[0054] n, p, r, s and t are each independently either 0 or 1.
[0055] As used herein, it is to be understood that the term folate
refers both individually to folic acid used in forming a conjugate,
or alternatively to a folate analog or derivative thereof that is
capable of binding to folate or folic acid receptors.
[0056] In one aspect of such folate analogs, when s is 1, t is 0,
and when s is 0, t is 1. In another aspect of such folate analogs,
both n and r are 1, and linker L.sup.a is a naturally occurring
amino acid covalently linked to A.sup.2 at its alpha-amino group
through an amide bond. Illustrative amino acids include aspartic
acid, glutamic acid, lysine, cysteine, and the like.
[0057] The vitamin can be folate which includes a nitrogen, and in
this embodiment, the spacer linkers can be alkylenecarbonyl,
cycloalkylenecarbonyl, carbonylalkylcarbonyl,
1-alkylenesuccinimid-3-yl, 1-(carbonytalkyl)succinimid-3-yl,
wherein each of the spacer linkers is optionally substituted with a
substituent X.sup.1, and the spacer linker is bonded to the folate
nitrogen to form an imide or an alkylamide. In this embodiment, the
substituents X.sup.1 can be alkyl, hydroxyalkyl, amino, aminoalkyl,
alkylaminoalkyl, dialkyaminoalkyl, sulfhydrylalkyl, alkylthioalkyl,
aryl, substituted aryl, arylalkyl, substituted arylalkyl, carboxy,
carboxyalkyl, guanidinoalkyl, R.sup.4-carbonyl,
R.sup.5-carbonylalkyl, R.sup.6-acylamino, and
R.sup.7-acylaminoalkyl, wherein R.sup.4 and R.sup.5 are each
independently selected from amino acids, amino acid derivatives,
and peptides, and wherein R.sup.6 and R.sup.7 are each
independently selected from amino acids, amino acid derivatives,
and peptides.
[0058] Illustrative embodiments of vitamin analogs and/or
derivatives also include analogs and derivatives of biotin such as
biocytin, biotin sulfoxide, oxybiotin and other biotin
receptor-binding compounds, and the like. It is appreciated that
analogs and derivatives of the other vitamins described herein are
also contemplated herein. In one embodiment, vitamins that can be
used as the binding ligand (B) in the drug delivery conjugates
described herein include those that bind to vitamin receptors
expressed specifically on activated macrophages, such as the folate
receptor, which binds folate, or an analog or derivative thereof as
described herein.
[0059] In addition to the vitamins described herein, it is
appreciated that other binding ligands may be coupled with the
drugs and linkers described and contemplated herein to form binding
ligand-linker-drug conjugates capable of facilitating delivery of
the drug to a desired target. These other binding ligands, in
addition to the vitamins and their analogs and derivatives
described, may be used to form drug delivery conjugates capable of
binding to target cells. In general, any binding ligand (B) of a
cell surface receptor may be advantageously used as a targeting
ligand to which a linker-drug conjugate can be attached.
[0060] Illustrative other ligands described herein include peptide
ligands identified from library screens, tumor cell-specific
peptides, tumor cell-specific aptamers, tumor cell-specific
carbohydrates, tumor cell-specific monoclonal or polyclonal
antibodies, Fab or scFv (i.e., a single chain variable region)
fragments of antibodies such as, for example, an Fab fragment of an
antibody directed to EphA2 or other proteins specifically expressed
or uniquely accessible on metastatic cancer cells, small organic
molecules derived from combinatorial libraries, growth factors,
such as EGF, FGF, insulin, and insulin-like growth factors, and
homologous polypeptides, somatostatin and its analogs, transferrin,
lipoprotein complexes, bile salts, selectins, steroid hormones,
Arg-Gly-Asp containing peptides, retinoids, various Galectins,
.delta.-opioid receptor ligands, cholecystokinin A receptor
ligands, ligands specific for angiotensin AT1 or AT2 receptors,
peroxisome proliferator-activated receptor .lamda. ligands,
.beta.-lactam antibiotics such as penicillin, small organic
molecules including antimicrobial drugs, and other molecules that
bind specifically to a receptor preferentially expressed on the
surface of tumor cells or on an infectious organism, antimicrobial
and other drugs designed to fit into the binding pocket of a
particular receptor based on the crystal structure of the receptor
or other cell surface protein, binding ligands of tumor antigens or
other molecules preferentially expressed on the surface of tumor
cells, or fragments of any of these molecules.
[0061] An example of a tumor-specific antigen that could function
as a binding site for a binding ligand-drug conjugate include
extracellular epitopes of a member of the Ephrin family of
proteins, such as EphA2. EphA2 expression is restricted to
cell-cell junctions in normal cells, but EphA2 is distributed over
the entire cell surface in metastatic tumor cells. Thus, EphA2 on
metastatic cells would be accessible for binding to, for example,
an Fab fragment of an antibody conjugated to a drug, whereas the
protein would not be accessible for binding to the Fab fragment on
normal cells, resulting in a binding ligand-drug conjugate specific
for metastatic cancer cells.
[0062] The linker L includes one or more hydrophilic spacer
linkers. In addition, other optional spacer linkers and/or
releasable linkers may be included in L. It is appreciated that
additional spacer linkers may included when predetermined lengths
are selccted for separating binding ligand B from agent A. It is
also appreciated that in certain configurations, releasable linkers
may be included. For example, as described herein in one
embodiment, the targeted ligand conjugates may be used to deliver
drugs for treating cancer or other diseases involving pathogenic
cells. In such embodiments, it is appreciated that once delivered,
the drug is desirably released from the conjugate. For example, in
the configuration where the targeting ligand is folate, or an
analog or derivative thereof, the conjugate may bind to a folate
receptor. Once bound, the conjugate often undergoes the process of
endocytosis, and the conjugate is delivered to the interior of the
cell. Cellular mechanisms may biologically degrade the conjugate to
release the drug "payload" and release the folate compound.
[0063] In an alternative configuration, the targeted conjugate may
be used in immunotherapy. In this configuration, a releasable
linker is generally not included. For example, conjugates of folate
or other vitamin receptor binding compounds and immunogens, once
delivered, will bind to the appropriated receptor and decorate or
mark the cell with the antigenic payload. In another alternative
configuration, the targeted conjugate may be used in a diagnostic
therapy. In this configuration, a releasable linker may or may not
be included. For example, conjugates that include imaging agents
may be delivered to a target cell using the appropriate cell
receptor binding ligand, such as a folate or other vitamin receptor
binding compound. In one aspect, the conjugate may remain on the
surface of the cell for imaging. In another configuration, the
conjugate may undergo endocytosis into the interior of the cell. In
this latter situation, a releasable linker may be included.
[0064] Accordingly, in other aspects, the conjugates B-L-A
described herein also include the following general formulae:
B-L.sub.S-L.sub.H-A
B-L.sub.H-L.sub.S-A
B-L.sub.S-L.sub.H-L.sub.S-A
B-L.sub.R-L.sub.H-A
B-L.sub.H-L.sub.R-A
B-L.sub.R-L.sub.H-L.sub.R-A
B-L.sub.S-L.sub.R-L.sub.H-A
B-L.sub.R-L.sub.H-L.sub.S-A
B-L.sub.R-L.sub.S-L.sub.H-L.sub.R-A
B-L.sub.H-L.sub.S-L.sub.H-L.sub.R-A
where B, L, and A are as described herein, and L.sub.R is a
releasable linker section, L.sub.S is a spacer linker section, and
L.sub.H is a hydrophilic linker section of linker L. It is to be
understood that the foregoing formulae are merely illustrative, and
that other arrangements of the hydrophilic spacer linker sections,
releasable linker sections, and spacer linker sections are to be
included herein. In addition, it is to be understood that
additional conjugates are contemplated that include a plurality
hydrophilic spacer linkers, and/or a plurality of releasable
linkers, and/or a plurality of spacer linkers.
[0065] Similarly, in other aspects, the conjugates L-A described
herein also include the following general formulae:
L.sub.S-L.sub.H-A
L.sub.H-L.sub.S-A
L.sub.S-L.sub.H-L.sub.S-A
L.sub.R-L.sub.H-A
L.sub.H-L.sub.R-A
L.sub.R-L.sub.H-L.sub.R-A
L.sub.S-L.sub.R-L.sub.H-A
L.sub.R-L.sub.H-L.sub.S-A
L.sub.R-L.sub.S-L.sub.H-L.sub.R-A
L.sub.H-L.sub.S-L.sub.H-L.sub.R-A
where L and A are as described herein, and L.sub.R is a releasable
linker section, L.sub.S is a spacer linker section, and L.sub.H is
a hydrophilic tinker section of linker L. It is to be understood
that the foregoing formulae are merely illustrative, and that other
arrangements of the hydrophilic spacer linker sections, releasable
linker sections, and spacer linker sections are to be included
herein. In addition, it is to be understood that additional
conjugates are contemplated that include a plurality hydrophilic
spacer linkers, and/or a plurality of releasable linkers, and/or a
plurality of spacer linkers.
[0066] It is appreciated that the arrangement and/or orientation of
the various hydrophilic linkers may be in a linear or branched
fashion, or both. For example, the hydrophilic linkers may form the
backbone of the linker forming the conjugate between the folate and
the drug, imagining agent, or diagnostic agent. Alternatively, the
hydrophilic portion of the linker may be pendant to or attached to
the backbone of the chain of atoms connecting the binding ligand B
to the agent A. In this latter arrangement, the hydrophilic portion
may be proximal or distal to the backbone chain of atoms.
[0067] In another embodiment, the linker is more or less linear,
and the hydrophilic groups are arranged largely in a series to form
a chain-like linker in the conjugate. Said another way, the
hydrophilic groups form some or all of the backbone of the linker
in this linear embodiment.
[0068] In another embodiment, the linker is branched with
hydrophilic groups. In this branched embodiment, the hydrophilic
groups may be proximal to the backbone or distal to the backbone.
In each of these arrangements, the linker is more spherical or
cylindrical in shape. In one variation, the linker is shaped like a
bottle-brush. In one aspect, the backbone of the linker is formed
by a linear series of amides, and the hydrophilic portion of the
linker is formed by a parallel arrangement of branching side
chains, such as by connecting monosaccharides, sulfonates, and the
like, and derivatives and analogs thereof.
[0069] It is understood that the linker may be neutral or ionizable
under certain conditions, such as physiological conditions
encountered in vivo. For ionizable linkers, under the selected
conditions, the linker may deprotonate to form a negative ion, or
alternatively become protonated to form a positive ion. It is
appreciated that more than one deprotonation or protonation event
may occur. In addition, it is understood that the same linker may
deprotonate and protonate to form inner salts or zwitterionic
compounds.
[0070] In another embodiment, the hydrophilic spacer linkers are
neutral, i.e. under physiological conditions, the linkers do not
significantly protonate nor deprotonate. In another embodiment, the
hydrophilic spacer linkers may be protonated to carry one or more
positive charges. It is understood that the protonation capability
is condition dependent. In one aspect, the conditions are
physiological conditions, and the linker is protonated in vivo. In
another embodiment, the spacers include both regions that are
neutral and regions that may be protonated to carry one or more
positive charges. In another embodiment, the spacers include both
regions that may be deprotonated to carry one or more negative
charges and regions that may be protonated to carry one or more
positive charges. It is understood that in this latter embodiment
that zwitterions or inner salts may be formed.
[0071] In one aspect, the regions of the linkers that may be
deprotonated to carry a negative charge include carboxylic acids,
such as aspartic acid, glutamic acid, and longer chain carboxylic
acid groups, and sulfuric acid esters, such as alkyl esters of
sulfuric acid. In another aspect, the regions of the linkers that
may be protonated to carry a positive charge include amino groups,
such as polyaminoalkylenes including ethylene diamines, propylene
diamines, butylene diamines and the like, and/or heterocycles
including pyrollidines, piperidines, piperazines, and other amino
groups, each of which is optionally substituted. In another
embodiment, the regions of the linkers that are neutral include
poly hydroxyl groups, such as sugars, carbohydrates, saccharides,
inositols, and the like, and/or polyether groups, such as
polyoxyalkylene groups including polyoxyethylene, polyoxypropylene,
and the like.
[0072] In one embodiment, the hydrophilic spacer linkers described
herein include are formed primarily from carbon, hydrogen, and
oxygen, and have a carbon/oxygen ratio of about 3:1 or less, or of
about 2:1 or less. In one aspect, the hydrophilic linkers described
herein include a plurality of ether functional groups. In another
aspect, the hydrophilic linkers described herein include a
plurality of hydroxyl functional groups. Illustrative fragments
that may be used to form such linkers include polyhydroxyl
compounds such as carbohydrates, polyether compounds such as
polyethylene glycol units, and acid groups such as carboxyl and
alkyl sulfuric acids. In one variation, oligoamide spacers, and the
like may also be included in the linker.
[0073] Illustrative carbohydrate spacers include saccharopeptides
as described herein that include both a peptide feature and sugar
feature; glucuronides, which may be incorporated via [2+3] Huisgen
cyclization, also known as click chemistry; .beta.-alkyl
glycosides, such as of 2-deoxyhexapyranoses (2-deoxyglucose,
2-deoxyglucuronide, and the like), and .beta.-alkyl
mannopyranosides. Illustrative PEG groups include those of a
specific length range from about 4 to about 20 PEG groups.
Illustrative alkyl sulfuric acid esters may also be introduced with
click chemistry directly into the backbone. Illustrative oligoamide
spacers include EDTA and DTPA spacers, .beta.-amino acids, and the
like.
[0074] In another embodiment, the hydrophilic spacer linkers
described herein include a polyether, such as the linkers of the
following formulae:
##STR00002##
where m is an integer independently selected in each instance from
1 to about 8; p is an integer selected 1 to about 10; and n is an
integer independently selected in each instance from 1 to about 3.
In one aspect, m is independently in each instance 1 to about 3. In
another aspect, n is 1 in each instance. In another aspect, p is
independently in each instance about 4 to about 6. Illustratively,
the corresponding polypropylene polyethers corresponding to the
foregoing are contemplated herein and may be included in the
conjugates as hydrophilic spacer linkers. In addition, it is
appreciated that mixed polyethylene and polypropylene polyethers
may be included in the conjugates as hydrophilic spacer linkers.
Further, cyclic variations of the foregoing polyether compounds,
such as those that include tetrahydrofuranyl, 1,3-dioxanes,
1,4-dioxanes, and the like are contemplated herein.
[0075] In another illustrative embodiment, the hydrophilic spacer
linkers described herein include a plurality of hydroxyl functional
groups, such as linkers that incorporate monosaccharides,
oligosaccharides, polysaccharides, and the like. It is to be
understood that the polyhydroxyl containing spacer linkers
comprises a plurality of --(CROH)-- groups, where R is hydrogen or
alkyl.
[0076] In another embodiment, the spacer linkers include one or
more of the following fragments:
##STR00003##
wherein R is H, alkyl, cycloalkyl, or arylalkyl; m is an integer
from 1 to about 3; n is an integer from 1 to about 5, p is an
integer from 1 to about 5, and r is an integer selected from 1 to
about 3. In one aspect, the integer n is 3 or 4. In another aspect,
the integer p is 3 or 4. In another aspect, the integer r is 1.
[0077] In another embodiment, the spacer linker includes one or
more of the following cyclic polyhydroxyl groups:
##STR00004## ##STR00005##
wherein n is an integer from 2 to about 5, p is an integer from 1
to about 5, and r is an integer from 1 to about 4. In one aspect,
the integer n is 3 or 4. In another aspect, the integer p is 3 or
4. In another aspect, the integer r is 2 or 3. It is understood
that all stereochemical forms of such sections of the linkers are
contemplated herein. For example, in the above formula, the section
may be derived from ribose, xylose, glucose, mannose, galactose, or
other sugar and retain the stereochemical arrangements of pendant
hydroxyl and alkyl groups present on those molecules. In addition,
it is to be understood that in the foregoing formulae, various
deoxy compounds are also contemplated. Illustratively, compounds of
the following formulae are contemplated:
##STR00006##
wherein n is equal to or less than r, such as when r is 2 or 3, n
is 1 or 2, or 1, 2, or 3, respectively.
[0078] In another embodiment, the spacer linker includes a
polyhydroxyl compound of the following formula:
##STR00007##
wherein n and r are each an integer selected from 1 to about 3. In
one aspect, the spacer linker includes one or more polyhydroxyl
compounds of the following formulae:
##STR00008##
[0079] It is understood that all stereochemical forms of such
sections of the linkers are contemplated herein. For example, in
the above formula, the section may be derived from ribose, xylose,
glucose, mannose, galactose, or other sugar and retain the
stereochemical arrangements of pendant hydroxyl and alkyl groups
present on those molecules.
[0080] In another configuration, the hydrophilic linkers L
described herein include polyhydroxyl groups that are spaced away
from the backbone of the linker. In one embodiment, such
carbohydrate groups or polyhydroxyl groups are connected to the
back bone by a triazole group, forming triazole-linked hydrophilic
spacer linkers. Illustratively, such linkers include fragments of
the following formulae:
##STR00009##
wherein n, m, and r are integers and are each independently
selected in each instance from 1 to about 5. In one illustrative
aspect, m is independently 2 or 3 in each instance. In another
aspect, r is 1 in each instance. In another aspect, n is 1 in each
instance. In one variation, the group connecting the polyhydroxyl
group to the backbone of the linker is a different heteroaryl
group, including but not limited to, pyrrole, pyrazole,
1,2,4-triazole, furan, oxazole, isoxazole, thienyl, thiazole,
isothiazole, oxadiazole, and the like. Similarly, divalent
6-membered ring heteroaryl groups are contemplated. Other
variations of the foregoing illustrative hydrophilic spacer linkers
include oxyalkylene groups, such as the following formulae:
##STR00010##
wherein n and r are integers and are each independently selected in
each instance from 1 to about 5; and p is an integer selected from
1 to about 4.
[0081] In another embodiment, such carbohydrate groups or
polyhydroxyl groups are connected to the back bone by an amide
group, forming amide-linked hydrophilic spacer linkers.
Illustratively, such linkers include fragments of the following
formulae:
##STR00011##
wherein n is an integer selected from 1 to about 3, and m is an
integer selected from 1 to about 22. In one illustrative aspect, n
is 1 or 2. In another illustrative aspect, m is selected from about
6 to about 10, illustratively 8. In one variation, the group
connecting the polyhydroxyl group to the backbone of the linker is
a different functional group, including but not limited to, esters,
ureas, carbamates, acylhydrazones, and the like. Similarly, cyclic
variations are contemplated. Other variations of the foregoing
illustrative hydrophilic spacer linkers include oxyalkylene groups,
such as the following formulae:
##STR00012##
wherein n and r are integers and are each independently selected in
each instance from 1 to about 5; and p is an integer selected from
1 to about 4.
[0082] In another embodiment the spacer linkers include one or more
of the following fragments:
##STR00013## ##STR00014##
wherein R is H, alkyl, cycloalkyl, or arylalkyl; m is an
independently selected integer from 1 to about 3; n is an integer
from 1 to about 6, p is an integer from 1 to about 5, and r is an
integer selected from 1 to about 3. In one variation, the integer n
is 3 or 4. In another variation, the integer p is 3 or 4. In
another variation, the integer r is 1.
[0083] In another embodiment, the spacer linkers include one or
more of the following fragments:
##STR00015## ##STR00016##
wherein m is an independently selected integer from 1 to about 3; n
is an integer from 1 to about 6, p is an integer from 1 to about 5,
and r is an integer selected from 1 to about 3. In one variation,
the integer n is 3 or 4. In another variation, the integer p is 3
or 4. In another variation, the integer r is 1.
[0084] In another embodiment, the spacer linkers include one or
more of the following fragments:
##STR00017## ##STR00018## ##STR00019##
wherein m is an independently selected integer from 1 to about 3; n
is an integer from 1 to about 6, p is an integer from 1 to about 5,
and r is an integer selected from 1 to about 3. In one variation,
the integer n is 3 or 4. In another variation, the integer p is 3
or 4. In another variation, the integer r is 1.
[0085] In another embodiment, the hydrophilic spacer linker is a
combination of backbone and branching side motifs such as is
illustrated by the following formulae
##STR00020##
wherein n is an integer independently selected in each instance
from 0 to about 3. The above formula are intended to represent 4,
5, 6, and even larger membered cyclic sugars. In addition, it is to
be understood that the above formula may be modified to represent
deoxy sugars, where one or more of the hydroxy groups present on
the formulae are replaced by hydrogen, alkyl, or amino. In
addition, it is to be understood that the corresponding carbonyl
compounds are contemplated by the above formulae, where one or more
of the hydroxyl groups is oxidized to the corresponding carbonyl.
In addition, in this illustrative embodiment, the pyranose includes
both carboxyl and amino functional groups and (a) can be inserted
into the backbone and (b) can provide synthetic handles for
branching side chains in variations of this embodiment. Any of the
pendant hydroxyl groups may be used to attach other chemical
fragments, including additional sugars to prepare the corresponding
oligosaccharides. Other variations of this embodiment are also
contemplated, including inserting the pyranose or other sugar into
the backbone at a single carbon, i.e. a spiro arrangement, at a
geminal pair of carbons, and like arrangements. For example, one or
two ends of the linker, or the agent A, or the binding ligand B may
be connected to the sugar to be inserted into the backbone in a
1,1; 1,2; 1,3; 1,4; 2,3, or other arrangement.
[0086] In another embodiment, the hydrophilic spacer linkers
described herein include are formed primarily from carbon,
hydrogen, and nitrogen, and have a carbon/nitrogen ratio of about
3:1 or less, or of about 2:1 or less. In one aspect, the
hydrophilic linkers described herein include a plurality of amino
functional groups.
[0087] In another embodiment, the spacer linkers include one or
more amino groups of the following formulae:
##STR00021##
where n is an integer independently selected in each instance from
1 to about 3. In one aspect, the integer n is independently 1 or 2
in each instance. In another aspect, the integer n is 1 in each
instance.
[0088] In another embodiment, the hydrophilic spacer linker is a
sulfuric acid ester, such as an alkyl ester of sulfuric acid.
Illustratively, the spacer linker is of the following formula:
##STR00022##
where n is an integer independently selected in each instance from
1 to about 3. Illustratively, n is independently 1 or 2 in each
instance.
[0089] It is understood, that in such polyhydroxyl, polyamino,
carboxylic acid, sulfuric acid, and like linkers that include free
hydrogens bound to heteroatoms, one or more of those free hydrogen
atoms may be protected with the appropriate hydroxyl, amino, or
acid protecting group, respectively, or alternatively may be
blocked as the corresponding pro-drugs, the latter of which are
selected for the particular use, such as pro-drugs that release the
parent drug under general or specific physiological conditions.
[0090] In each of the foregoing illustrative examples of linkers L,
there are also included in some cases additional spacer linkers
L.sub.S, and/or additional releasable linkers L.sub.R. Those spacer
linker and releasable linkers also may include asymmetric carbon
atoms. It is to be further understood that the stereochemical
configurations shown herein are merely illustrative, and other
stereochemical configurations are contemplated. For example in one
variation, the corresponding unnatural amino acid configurations
may be included in the conjugated described herein as follows:
##STR00023##
wherein n is an integer from 2 to about 5, p is an integer from 1
to about 5, and r is an integer from 1 to about 4, as described
above.
[0091] It is to be further understood that in the foregoing
embodiments, open positions, such as (*) atoms are locations for
attachment of the binding ligand (B) or the agent (A) to be
delivered. In addition, it is to be understood that such attachment
of either or both of B and A may be direct or through an
intervening linker. Intervening linkers include other spacer
linkers and/or releasable linkers. Illustrative additional spacer
linkers and releasable linkers that are included in the conjugated
described herein are described in U.S. patent application Ser. No.
10/765,335, the disclosure of which is incorporated herein by
reference.
[0092] In one embodiment, the hydrophilic spacer linker comprises
one or more carbohydrate containing or polyhydroxyl group
containing linkers. In another embodiment, the hydrophilic spacer
linker comprises at least three carbohydrate containing or
polyhydroxyl group containing linkers. In another embodiment, the
hydrophilic spacer linker comprises one or more carbohydrate
containing or polyhydroxyl group containing linkers, and one or
more aspartic acids. In another embodiment, the hydrophilic spacer
linker comprises one or more carbohydrate containing or
polyhydroxyl group containing linkers, and one or more glutamic
acids. In another embodiment, the hydrophilic spacer linker
comprises one or more carbohydrate containing or polyhydroxyl group
containing linkers, one or more glutamic acids, one or more
aspartic acids, and one or more beta amino alanines. In a series of
variations, in each of the foregoing embodiments, the hydrophilic
spacer linker also includes one or more cysteines. In another
series of variations, in each of the foregoing embodiments, the
hydrophilic spacer linker also includes at least one arginine.
[0093] In another embodiment, the hydrophilic spacer linker
comprises one or more divalent 1,4-piperazines that are included in
the chain of atoms connecting at least one of the binding ligands
(L) with at least one of the agents (A). In one variation, the
hydrophilic spacer linker includes one or more carbohydrate
containing or polyhydroxyl group containing linkers. In another
variation, the hydrophilic spacer linker includes one or more
carbohydrate containing or polyhydroxyl group containing linkers
and one or more aspartic acids. In another variation, the
hydrophilic spacer linker includes one or more carbohydrate
containing or polyhydroxyl group containing linkers and one or more
glutamic acids. In a series of variations, in each of the foregoing
embodiments, the hydrophilic spacer linker also includes one or
more cysteines. In another series of variations, in each of the
foregoing embodiments, the hydrophilic spacer linker also includes
at least one arginine.
[0094] In another embodiment, the hydrophilic spacer linker
comprises one or more oligoamide hydrophilic spacers, such as but
not limited to aminoethylpiperazinylacetamide.
[0095] In another embodiment, the hydrophilic spacer linker
comprises one or more triazole linked carbohydrate containing or
polyhydroxyl group containing linkers. In another embodiment, the
hydrophilic spacer linker comprises one or more amide linked
carbohydrate containing or polyhydroxyl group containing linkers.
In another embodiment, the hydrophilic spacer linker comprises one
or more PEG groups and one or more cysteines. In another
embodiment, the hydrophilic spacer linker comprises one or more
EDTE derivatives.
[0096] In another embodiment, the additional spacer linker can be
1-alkylenesuccinimid-3-yl, optionally substituted with a
substituent X.sup.1, as defined below, and the releasable linkers
can be methylene, 1-alkoxyalkylene, 1-alkoxycycloalkylene,
1-alkoxyalkylenecarbonyl, 1-alkoxycycloalkylenecarbonyl, wherein
each of the releasable linkers is optionally substituted with a
substituent X.sup.2, as defined below, and wherein the spacer
linker and the releasable linker are each bonded to the spacer
linker to form a succinimid-1-ylalkyl acetal or ketal.
[0097] The additional spacer linkers can be carbonyl,
thionocarbonyl, alkylene, cycloalkylene, alkylenecycloalkyl,
alkylenecarbonyl, cycloalkylenecarbonyl, carbonylalkylcarbonyl,
1-alkylenesuccinimid-3-yl, 1-(carbonylalkyl)succinimid-3-yl,
alkylenesulfoxyl, sulfonylalkyl, alkylenesulfoxylalkyl,
alkylenesulfonylalkyl, carbonyltetrahydro-2H-pyranyl,
carbonyltetrahydrofuranyl,
1-(carbonyltetrahydro-2H-pyranyl)succinimid-3-yl, and
1-(carbonyltetrahydrofuranyl)succinimid-3-yl, wherein each of the
spacer linkers is optionally substituted with a substituent
X.sup.1, as defined below. In this embodiment, the spacer linker
may include an additional nitrogen, and the spacer linkers can be
alkylenccarbonyl, cycloalkylenecarbonyl, carbonylalkylcarbonyl,
1-(carbonylalkyl)succinimid-3-yl, wherein each of the spacer
linkers is optionally substituted with a substituent X.sup.1, as
defined below, and the spacer linker is bonded to the nitrogen to
form an amide. Alternatively, the spacer linker may include an
additional sulfur, and the spacer linkers can be alkylene and
cycloalkylene, wherein each of the spacer linkers is optionally
substituted with carboxy, and the spacer linker is bonded to the
sulfur to form a thiol. In another embodiment, the spacer linker
can include sulfur, and the spacer linkers can be
1-alkylenesuccinimid-3-yl and 1-(carbonylalkyl)succinimid-3-yl, and
the spacer linker is bonded to the sulfur to form a
succinimid-3-ylthiol.
[0098] In an alternative to the above-described embodiments, the
additional spacer linker can include nitrogen, and the releasable
linker can be a divalent radical comprising alkyleneaziridin-1-yl,
carbonylalkylaziridin-1-yl, sulfoxylalkylaziridin-1-yl, or
sulfonylalkylaziridin-1-yl, wherein each of the releasable linkers
is optionally substituted with a substituent X.sup.2, as defined
below. In this alternative embodiment, the spacer linkers can be
carbonyl, thionocarbonyl, alkylenecarbonyl, cycloalkylenecarbonyl,
carbonylalkylcarbonyl, 1-(carbonylalkyl)succinimid-3-yl, wherein
each of the spacer linkers is optionally substituted with a
substituent X.sup.1, as defined below, and wherein the spacer
linker is bonded to the releasable linker to form an aziridine
amide.
[0099] The substituents X.sup.1 can be alkyl, alkoxy, alkoxyalkyl,
hydroxy, hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl,
dialkylaminoalkyl, halo, haloalkyl, sulfhydrylalkyl,
alkylthioalkyl, aryl, substituted aryl, arylalkyl, substituted
arylalkyl, heteroaryl, substituted heteroaryl, carboxy,
carboxyalkyl, alkyl carboxylate, alkyl alkanoate, guanidinoalkyl,
R.sup.4-carbonyl, R.sup.5-carbonylalkyl, R.sup.6-acylamino, and
R.sup.7-acylaminoalkyl, wherein R.sup.4 and R.sup.5 are each
independently selected from amino acids, amino acid derivatives,
and peptides, and wherein R.sup.6 and R.sup.7 are each
independently selected from amino acids, amino acid derivatives,
and peptides. In this embodiment the spacer linker can include
nitrogen, and the substituent X.sup.1 and the spacer linker to
which they are bound to form an heterocycle.
[0100] In another embodiment, the releasable linker may be a
divalent radical comprising alkyleneaziridin-1-yl,
alkylenecarbonylaziridin-1-yl, carbonylalkylaziridin-1-yl,
alkylenesulfoxylaziridin-1-yl, sulfoxylalkylaziridin-1-yl,
sulfonylalkylaziridin-1-yl, or alkylenesulfonylaziridin-1-yl,
wherein each of the releasable linkers is optionally substituted
with a substituent X.sup.2, as defined below.
[0101] Additional illustrative releasable linkers include
methylene, 1-alkoxyalkylene, 1-alkoxycycloalkylene,
1-alkoxyalkylenecarbonyl, 1-alkoxycycloalkylenecarbonyl,
carbonylarylcarbonyl, carbonyl(carboxyaryl)carbonyl,
carbonyl(biscarboxyaryl)carbonyl, haloalkylenecarbonyl,
alkylene(dialkylsilyl), alkylene(alkylarylsilyl),
alkylene(diarylsilyl), (dialkylsilyl)aryl, (alkylarylsilyl)aryl,
(diarylsilyl)aryl, oxycarbonyloxy, oxycarbonyloxyalkyl,
sulfonyloxy, oxysulfonylalkyl, iminoalkylidenyl,
carbonylalkylideniminyl, iminocycloalkylidenyl,
carbonylcycloalkylideniminyl, alkylenethio, alkylenearylthio, and
carbonylalkylthio, wherein each of the releasable linkers is
optionally substituted with a substituent X.sup.2, as defined
below.
[0102] In the preceding embodiment, the releasable linker may
include oxygen, and the releasable linkers can be methylene,
1-alkoxyalkylene, 1-alkoxycycloalkylene, 1-alkoxyalkylenecarbonyl,
and 1-alkoxycycloalkylenecarbonyl, wherein each of the releasable
linkers is optionally substituted with a substituent X.sup.2, as
defined below, and the releasable linker is bonded to the oxygen to
form an acetal or ketal. Alternatively, the releasable linker may
include oxygen, and the releasable linker can be methylene, wherein
the methylene is substituted with an optionally-substituted aryl,
and the releasable linker is bonded to the oxygen to form an acetal
or ketal. Further, the releasable linker may include oxygen, and
the releasable linker can be sulfonylalkyl, and the releasable
linker is bonded to the oxygen to form an alkylsulfonate.
[0103] In another embodiment of the above releasable linker
embodiment, the releasable linker may include nitrogen, and the
releasable linkers can be iminoalkylidenyl,
carbonylalkylideniminyl, iminocycloalkylidenyl, and
carbonylcycloalkylideniminyl, wherein each of the releasable
linkers is optionally substituted with a substituent X.sup.2, as
defined below, and the releasable linker is bonded to the nitrogen
to form an hydrazone. In an alternate configuration, the hydrazone
may be acylated with a carboxylic acid derivative, an orthoformate
derivative, or a carbamoyl derivative to form various acylhydrazone
releasable linkers.
[0104] Alternatively, the releasable linker may include oxygen, and
the releasable linkers can be alkylene(dialkylsilyl),
alkylene(alkylarylsilyl), alkylene(diarylsilyl),
(dialkylsilyl)aryl, (alkylarylsilyl)aryl, and (diarylsilyl)aryl,
wherein each of the releasable linkers is optionally substituted
with a substituent X.sup.2, as defined below, and the releasable
linker is bonded to the oxygen to form a silanol.
[0105] In the above releasable tinker embodiment, the drug can
include a nitrogen atom, the releasable linker may include
nitrogen, and the releasable linkers can be carbonylarylcarbonyl,
carbonyl(carboxyaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl,
and the releasable linker can be bonded to the heteroatom nitrogen
to form an amide, and also bonded to the drug nitrogen to form an
amide.
[0106] In the above releasable tinker embodiment, the drug can
include an oxygen atom, the releasable linker may include nitrogen,
and the releasable linkers can be carbonylarylcarbonyl,
carbonyl(carboxyaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl,
and the releasable linker can form an amide, and also bonded to the
drug oxygen to form an ester.
[0107] The substituents X.sup.2 can be alkyl, alkoxy, alkoxyakyl,
hydroxy, hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl,
dialkylaminoalkyl, halo, haloalkyl, sulfhydrylalkyl,
alkylthioalkyl, aryl, substituted aryl, arylalkyl, substituted
arylalkyl, heteroaryl, substituted heteroaryl, carboxy,
carboxyalkyl, alkyl carboxylate, alkyl alkanoate, guanidinoalkyl,
R.sup.4-carbonyl, R.sup.5-carbonylalkyl, R.sup.6-acylamino, and
R.sup.7-acylaminoalkyl, wherein R.sup.4 and R.sup.5 are each
independently selected from amino acids, amino acid derivatives,
and peptides, and wherein R.sup.6 and R.sup.7 are each
independently selected from amino acids, amino acid derivatives,
and peptides. In this embodiment the releasable linker can include
nitrogen, and the substituent X.sup.2 and the releasable linker can
form an heterocycle.
[0108] The heterocycles can be pyrrolidines, piperidines,
oxazolidines, isoxazolidines, thiazolidines, isothiazolidines,
pyrrolidinones, piperidinones, oxazolidinones, isoxazolidinones,
thiazolidinones, isothiazolidinones, and succinimides.
[0109] The agent A can include a nitrogen atom, and the releasable
linker can be haloalkylenecarbonyl, optionally substituted with a
substituent X.sup.2, and the releasable linker is bonded to the
drug nitrogen to form an amide.
[0110] The agent A can include an oxygen atom, and the releasable
linker can be haloalkylenecarbonyl, optionally substituted with a
substituent X.sup.2, and the releasable linker is bonded to the
drug oxygen to form an ester.
[0111] The agent A can include a double-bonded nitrogen atom, and
in this embodiment, the releasable linkers can be
alkylenecarbonylamino and 1-(alkylenecarbonylamino)succinimid-3-yl,
and the releasable linker can be bonded to the drug nitrogen to
form an hydrazone.
[0112] The agent A can include a sulfur atom, and in this
embodiment, the releasable linkers can be alkylenethio and
carbonylalkylthio, and the releasable linker can be bonded to the
drug sulfur to form a disulfide.
[0113] The agent A can be a mitomycin, a mitomycin derivative, or a
mitomycin analog, and, in this embodiment, the releasable linkers
can be carbonylalkylthio, carbonyltetrahydro-2H-pyranyl,
carbonyltetrahydrofuranyl,
1-(carbonyltetrahydro-2H-pyranyl)succinimid-3-yl, and
1-(carbonyltetrahydrofuranyl)succinimid-3-yl, wherein each of the
releasable linkers is optionally substituted with a substituent
X.sup.2, and wherein the aziridine of the mitomycin is bonded to
the releasable linker to form an acylaziridine.
[0114] The binding ligand B can be folate which includes a
nitrogen, and in this embodiment, the spacer linkers can be
alkylenecarbonyl, cycloalkylenecarbonyl, carbonylalkylcarbonyl,
1-alkylenesuccinimid-3-yl, 1-(carbonylalkyl)succinimid-3-yl,
wherein each of the spacer linkers is optionally substituted with a
substituent X.sup.1, and the spacer linker is bonded to the folate
nitrogen to form an imide or an alkylamide. In this embodiment, the
substituents X.sup.1 can be alkyl, hydroxyalkyl, amino, aminoalkyl,
alkylaminoalkyl, dialkylaminoalkyl, sulfhydrylalkyl,
alkylthioalkyl, aryl, substituted aryl, arylalkyl, substituted
arylalkyl, carboxy, carboxyalkyl, guanidinoalkyl, R.sup.4-carbonyl,
R.sup.5-carbonylalkyl, R.sup.6-acylamino, and
R.sup.7-acylaminoalkyl, wherein R.sup.4 and R.sup.5 are each
independently selected from amino acids, amino acid derivatives,
and peptides, and wherein R.sup.6 and R.sup.7 are each
independently selected from amino acids, amino acid derivatives,
and peptides.
[0115] The term cycloalkylene as used herein refers to a bivalent
chain of carbon atoms, a portion of which forms a ring, such as
cycloprop-1,1-diyl, cycloprop-1,2-diyl, cyclohex-1,4-diyl,
3-ethylcyclopent-1,2-diyl, 1-methylenecyclohex-4-yl, and the
like.
[0116] The term heterocycle as used herein refers to a monovalent
chain of carbon and heteroatoms, wherein the heteroatoms are
selected from nitrogen, oxygen, and sulfur, a portion of which,
including at least one heteroatom, form a ring, such as aziridine,
pyrrolidine, oxazolidine, 3-methoxypyrrolidine, 3-methylpiperazine,
and the like.
[0117] The term aryl as used herein refers to an aromatic mono or
polycyclic ring of carbon atoms, such as phenyl, naphthyl, and the
like. In addition, aryl may also include heteroaryl.
[0118] The term heteroaryl as used herein refers to an aromatic
mono or polycyclic ring of carbon atoms and at least one heteroatom
selected from nitrogen, oxygen, and sulfur, such as pyridinyl,
pyrimidinyl, indolyl, benzoxazolyl, and the like.
[0119] The term optionally substituted as used herein refers to the
replacement of one or more hydrogen atoms, generally on carbon,
with a corresponding number of substituents, such as halo, hydroxy,
amino, alkyl or dialkylamino, alkoxy, alkylsulfonyl, cyano, nitro,
and the like. In addition, two hydrogens on the same carbon, on
adjacent carbons, or nearby carbons may be replaced with a bivalent
substituent to form the corresponding cyclic structure.
[0120] The term iminoalkylidenyl as used herein refers to a
divalent radical containing alkylene as defined herein and a
nitrogen atom, where the terminal carbon of the alkylene is
double-bonded to the nitrogen atom, such as the formulae
--(CH).dbd.N--, --(CH.sub.2).sub.2(CH).dbd.N--,
--CH.sub.2C(Me).dbd.N--, and the like.
[0121] The term amino acid as used herein refers generally to
aminoalkylcarboxylate, where the alkyl radical is optionally
substituted, such as with alkyl, hydroxy alkyl, sulfhydrylalkyl,
aminoalkyl, carboxyalkyl, and the like, including groups
corresponding to the naturally occurring amino acids, such as
scrine, cysteine, methionine, aspartic acid, glutamic acid, and the
like. It is to be understood that such amino acids may be of a
single stereochemistry or a particular mixture of stereochemisties,
including racemic mixtures. In addition, amino acid refers to beta,
gamma, and longer amino acids, such as amino acids of the
formula:
--N(R)--(CR'R'').sub.qC(O)--
where R is hydrogen, alkyl, acyl, or a suitable nitrogen protecting
group, R' and R'' are hydrogen or a substituent, each of which is
independently selected in each occurrence, and q is an integer such
as 1, 2, 3, 4, or 5. Illustratively, R' and/or R'' independently
correspond to, but are not limited to, hydrogen or the side chains
present on naturally occurring amino acids, such as methyl, benzyl,
hydroxymethyl, thiomethyl, carboxyl, carboxylmethyl,
guanidinopropyl, and the like, and derivatives and protected
derivatives thereof. The above described formula includes all
stereoisomeric variations. For example, the amino acid may be
selected from asparagine, aspartic acid, cysteine, glutamic acid,
lysine, glutamine, arginine, serine, ornitine, threonine, and the
like. In another illustrative aspect of the vitamin receptor
binding drug delivery conjugate intermediate described herein, the
drug, or an analog or a derivative thereof, includes an alkylthiol
nucleophile.
[0122] It is to be understood that the above-described terms can be
combined to generate chemically-relevant groups, such as
alkoxyalkyl referring to methyloxymethyl, ethyloxyethyl, and the
like, haloalkoxyalkyl referring to trifluoromethyloxyethyl,
1,2-difluoro-2-chloroeth-1-yloxypropyl, and the like, arylalkyl
referring to benzyl, phenethyl, .alpha.-methylbenzyl, and the like,
and others.
[0123] The term amino acid derivative as used herein refers
generally to an optionally substituted aminoalkylcarboxylate, where
the amino group and/or the carboxylate group are each optionally
substituted, such as with alkyl, carboxylalkyl, alkylamino, and the
like, or optionally protected. In addition, the optionally
substituted intervening divalent alkyl fragment may include
additional groups, such as protecting groups, and the like.
[0124] The term peptide as used herein refers generally to a series
of amino acids and/or amino acid analogs and derivatives covalently
linked one to the other by amide bonds.
[0125] The term "releasable linker" as used herein refers to a
linker that includes at least one bond that can be broken under
physiological conditions (e.g., a pH-labile, acid-labile,
oxidatively-labile, or enzyme-labile bond). It should be
appreciated that such physiological conditions resulting in bond
breaking include standard chemical hydrolysis reactions that occur,
for example, at physiological pH, or as a result of
compartmentalization into a cellular organelle such as an endosome
having a lower pH than cytosolic pH.
[0126] The cleavable bond or bonds may be present in the interior
of a cleavable linker and/or at one or both ends of a cleavable
linker. It is appreciated that the lability of the cleavable bond
may be adjusted by including functional groups or fragments within
the polyvalent linker L that are able to assist or facilitate such
bond breakage, also termed anchimeric assistance. In addition, it
is appreciated that additional functional groups or fragments may
be included within the polyvalent linker L that are able to assist
or facilitate additional fragmentation of the receptor binding
ligand agent conjugates after bond breaking of the releasable
linker. The lability of the cleavable bond can be adjusted by, for
example, substitutional changes at or near the cleavable bond, such
as including alpha branching adjacent to a cleavable disulfide
bond, increasing the hydrophobicity of substituents on silicon in a
moiety having a silicon-oxygen bond that may be hydrolyzed,
homologating alkoxy groups that form part of a ketal or acetal that
may be hydrolyzed, and the like.
[0127] It is understood that a cleavable bond can connect two
adjacent atoms within the releasable linker and/or connect other
linkers or V and/or D, as described herein, at either or both ends
of the releasable linker. In the case where a cleavable bond
connects two adjacent atoms within the releasable linker, following
breakage of the bond, the releasable linker is broken into two or
more fragments. Alternatively, in the case where a cleavable bond
is between the releasable linker and another moiety, such as an
additional heteroatom, additional spacer linker, another releasable
linker, the agent A, or analog or derivative thereof, or the
binding ligand B. or analog or derivative thereof, following
breakage of the bond, the releasable linker is separated from the
other moiety.
[0128] It is understood that each of the additional spacer and
releasable linkers are bivalent. It should be further understood
that the connectivity between each of the various additional spacer
and releasable linkers themselves, and between the various
additional spacer and releasable linkers and A and/or B, as defined
herein, may occur at any atom found in the various additional
spacer or releasable linkers.
[0129] In one aspect of the various receptor binding drug delivery
conjugates described herein, the polyvalent linker comprises an
additional spacer linker and a releasable linker taken together to
form 3-thiosuccinimid-1-ylalkyloxymethyloxy, where the methyl is
optionally substituted with alkyl or substituted aryl.
[0130] In another aspect, the polyvalent linker comprises an
additional spacer linker and a releasable linker taken together to
form 3-thiosuccinimid-1-ylalkylcarbonyl, where the carbonyl forms
an acylaziridine with the agent A, or analog or derivative
thereof.
[0131] In another aspect, the polyvalent linker comprises an
additional spacer linker and a releasable linker taken together to
form 1-alkoxycycloalkylenoxy.
[0132] In another aspect, the polyvalent linker comprises an
additional spacer linker and a releasable linker taken together to
form alkyleneaminocarbonyl(dicarboxylarylene)carboxylate.
[0133] In another aspect, the polyvalent linker comprises a
releasable linker, an additional spacer linker, and a releasable
linker taken together to form dithioalkylcarbonylhydrazide, where
the hydrazide forms an hydrazone with the agent A, or analog or
derivative thereof.
[0134] In another aspect, the polyvalent linker comprises an
additional spacer linker and a releasable linker taken together to
form 3-thiosuccinimid-1-ylalkylcarbonylhydrazide, where the
hydrazide forms an hydrazone with the agent A, or analog or
derivative thereof.
[0135] In another aspect, the polyvalent linker comprises an
additional spacer linker and a releasable linker taken together to
form 3-thioalkylsulfonylalkyl(disubstituted silyl)oxy, where the
disubstituted silyl is substituted with alkyl or optionally
substituted aryl.
[0136] In another aspect, the polyvalent linker comprises a
plurality of additional spacer linkers selected from the group
consisting of the naturally occurring amino acids and stereoisomers
thereof.
[0137] In another aspect, the polyvalent linker comprises a
releasable linker, an additional spacer linker, and a releasable
linker taken together to form 3-dithioalkyloxycarbonyl, where the
carbonyl forms a carbonate with the agent A, or analog or
derivative thereof.
[0138] In another aspect, the polyvalent linker comprises a
releasable linker, an additional spacer linker, and a releasable
linker taken together to form 3-dithioarylalkyloxycarbonyl, where
the carbonyl forms a carbonate with the agent A, or analog or
derivative thereof, and the aryl is optionally substituted.
[0139] In another aspect, the polyvalent linker comprises an
additional spacer linker and a releasable linker taken together to
form 3-thiosuccinimid-1-ylalkyloxyalkyloxyalkylidene, where the
alkylidene forms an hydrazone with the agent A, or analog or
derivative thereof, each alkyl is independently selected, and the
oxyalkyloxy is optionally substituted with alkyl or optionally
substituted aryl.
[0140] In another aspect, the polyvalent linker comprises a
releasable linker, an additional spacer linker, and a releasable
linker taken together to form
3-dithioalkyloxycarbonylhydrazide.
[0141] In another aspect, the polyvalent linker comprises a
releasable linker, an additional spacer linker, and a releasable
linker taken together to form 3-dithioalkylamino, where the amino
forms a vinylogous amide with the agent A, or analog or derivative
thereof.
[0142] In another aspect, the polyvalent linker comprises a
releasable linker, an additional spacer linker, and a releasable
linker taken together to form 3-dithioalkylamino, where the amino
forms a vinylogous amide with the agent A, or analog or derivative
thereof, and the alkyl is ethyl.
[0143] In another aspect, the polyvalent linker comprises a
releasable linker, an additional spacer linker, and a releasable
linker taken together to form 3-dithioalkylaminocarbonyl, where the
carbonyl forms a carbamate with the agent A, or analog or
derivative thereof.
[0144] In another aspect, the polyvalent linker comprises a
releasable linker, an additional spacer linker, and a releasable
linker taken together to form 3-dithioalkylaminocarbonyl, where the
carbonyl forms a carbamate with the agent A, or analog or
derivative thereof, and the alkyl is ethyl.
[0145] In another aspect, the polyvalent linker comprises a
releasable linker, an additional spacer linker, and a releasable
linker taken together to form 3-dithioarylalkyloxycarbonyl, where
the carbonyl forms a carbamate or a carbamoylaziridine with the
agent A, or analog or derivative thereof.
[0146] In another embodiment, the polyvalent linker (L) includes a
disulfide releasable linker. In another embodiment, the polyvalent
linker (L) includes at least one releasable linker that is not a
disulfide releasable linker.
[0147] In one aspect, the releasable and spacer linkers may be
arranged in such a way that subsequent to the cleavage of a bond in
the polyvalent linker, released functional groups chemically assist
the breakage or cleavage of additional bonds, also termed
anchimeric assisted cleavage or breakage. An illustrative
embodiment of such a polyvalent linker or portion thereof includes
compounds having the formulae:
##STR00024##
where X is an heteroatom, such as nitrogen, oxygen, or sulfur, or a
carbonyl group; n is an integer selected from 0 to 4;
illustratively 2; R is hydrogen, or a substituent, including a
substituent capable of stabilizing a positive charge inductively or
by resonance on the aryl ring, such as alkoxy and the like,
including methoxy; and the symbol (*) indicates points of
attachment for additional spacer, heteroatom, or releasable linkers
forming the polyvalent linker, or alternatively for attachment of
the drug, or analog or derivative thereof, or the vitamin, or
analog or derivative thereof. In one embodiment, n is 2 and R is
methoxy. It is appreciated that other substituents may be present
on the aryl ring, the benzyl carbon, the alkanoic acid, or the
methylene bridge, including but not limited to hydroxy, alkyl,
alkoxy, alkylthio, halo, and the like. Assisted cleavage may
include mechanisms involving benzylium intermediates, benzyne
intermediates, lactone cyclization, oxoniun intermediates,
beta-elimination, and the like. It is further appreciated that, in
addition to fragmentation subsequent to cleavage of the releasable
linker, the initial cleavage of the releasable linker may be
facilitated by an anchimerically assisted mechanism.
[0148] Illustrative examples of intermediates useful in forming
such linkers include:
##STR00025##
where X.sup.a is an electrophilic group such as maleimide, vinyl
sulfone, activated carboxylic acid derivatives, and the like,
X.sup.b is NH, O, or S; and m and n are each independently selected
integers from 0-4. In one variation, m and n are each independently
selected integers from 0-2. Such intermediates may be coupled to
drugs, binding ligands, or other linkers vai nucleophilic attack
onto electrophilic group X.sup.a, and/or by forming ethers or
carboxylic acid derivatives of the. In one embodiment, the benzylic
hydroxyl group is converted into the corresponding activated
benzyloxycarbonyl compound with phosgene or a phosgene equivalent.
This embodiment may be coupled to drugs, binding ligands, or other
linkers vai nucleophilic attack onto the activated carbonyl
group.
[0149] Illustrative mechanisms for cleavage of the bivalant linkers
described herein include the following 1,4 and 1,6 fragmentation
mechanisms
##STR00026##
where X is an exogenous or endogenous nucleophile, glutathione, or
bioreducing agent, and the like, and either of Z or Z' is the
vitamin, or analog or derivative thereof, or the drug, or analog or
derivative thereof, or a vitamin or drug moiety in conjunction with
other portions of the polyvalent linker. It is to be understood
that although the above fragmentation mechanisms are depicted as
concerted mechanisms, any number of discrete steps may take place
to effect the ultimate fragmentation of the polyvalent linker to
the final products shown. For example, it is appreciated that the
bond cleavage may also occur by acid-catalyzed elimination of the
carbamate moiety, which may be anchimerically assisted by the
stabilization provided by either the aryl group of the beta sulfur
or disulfide illustrated in the above examples. In those variations
of this embodiment, the releasable linker is the carbamate moiety.
Alternatively, the fragmentation may be initiated by a nucleophilic
attack on the disulfide group, causing cleavage to form a thiolate.
The thiolate may intermolecularly displace a carbonic acid or
carbamic acid moiety and form the corresponding thiacyclopropane.
In the case of the benzyl-containing polyvalent linkers, following
an illustrative breaking of the disulfide bond, the resulting
phenyl thiolate may further fragment to release a carbonic acid or
carbamic acid moiety by forming a resonance stabilized
intermediate. In any of these cases, the releasable nature of the
illustrative polyvalent linkers described herein may be realized by
whatever mechanism may be relevant to the chemical, metabolic,
physiological, or biological conditions present.
[0150] Other illustrative mechanisms for bond cleavage of the
releasable linker include oxonium-assisted cleavage as follows:
##STR00027##
where Z is the vitamin, or analog or derivative thereof, or the
drug, or analog or derivative thereof, or each is a vitamin or drug
moiety in conjunction with other portions of the polyvalent linker,
such as a drug or vitamin moiety including one or more spacer
linkers and/or other releasable linkers. Without being bound by
theory, in this embodiment, acid catalysis, such as in an endosome,
may initiate the cleavage via protonation of the urethane group. In
addition, acid-catalyzed elimination of the carbamate leads to the
release of CO.sub.2 and the nitrogen-containing moiety attached to
Z, and the formation of a benzyl cation, which may be trapped by
water, or any other Lewis base.
[0151] Other illustrative linkers include compounds of the
formulae:
##STR00028##
where X is NH, CH.sub.2, or O; R is hydrogen, or a substituent,
including a substituent capable of stabilizing a positive charge
inductively or by resonance on the aryl ring, such as alkoxy and
the like, including methoxy; and the symbol (*) indicates points of
attachment for additional spacer, heteroatom, or releasable linkers
forming the polyvalent linker, or alternatively for attachment of
the drug, or analog or derivative thereof, or the vitamin, or
analog or derivative thereof.
[0152] Illustrative mechanisms for cleavage of such polyvalent
linkers described herein include the following 1,4 and 1,6
fragmentation mechanisms followed by anchimerically assisted
cleavage of the acylated Z' via cyclization by the hydrazide
group:
##STR00029##
where X is an exogenous or endogenous nucleophile, glutathione, or
bioreducing agent, and the like, and either of Z or Z' is the
vitamin, or analog or derivative thereof, or the drug, or analog or
derivative thereof, or a vitamin or drug moiety in conjunction with
other portions of the polyvalent linker. It is to be understood
that although the above fragmentation mechanisms are depicted as
concerted mechanisms, any number of discrete steps may take place
to effect the ultimate fragmentation of the polyvalent linker to
the final products shown. For example, it is appreciated that the
bond cleavage may also occur by acid-catalyzed elimination of the
carbamate moiety, which may be anchimerically assisted by the
stabilization provided by either the aryl group of the beta sulfur
or disulfide illustrated in the above examples. In those variations
of this embodiment, the releasable linker is the carbamate moiety.
Alternatively, the fragmentation may be initiated by a nucleophilic
attack on the disulfide group, causing cleavage to form a thiolate.
The thiolate may intermolecularly displace a carbonic acid or
carbamic acid moiety and form the corresponding thiacyclopropane.
In the case of the benzyl-containing polyvalent linkers, following
an illustrative breaking of the disulfide bond, the resulting
phenyl thiolate may further fragment to release a carbonic acid or
carbamic acid moiety by forming a resonance stabilized
intermediate. In any of these cases, the releasable nature of the
illustrative polyvalent linkers described herein may be realized by
whatever mechanism may be relevant to the chemical, metabolic,
physiological, or biological conditions present. Without being
bound by theory, in this embodiment, acid catalysis, such as in an
endosome, may also initiate the cleavage via protonation of the
urethane group. In addition, acid-catalyzed elimination of the
carbamate leads to the release of CO.sub.2 and the
nitrogen-containing moiety attached to Z, and the formation of a
benzyl cation, which may be trapped by water, or any other Lewis
base, as is similarly described herein.
[0153] In one embodiment, the polyvalent linkers described herein
are compounds of the following formulae
##STR00030##
where n is an integer selected from 1 to about 4; R.sup.a and R are
each independently selected from the group consisting of hydrogen
and alkyl, including lower alkyl such as C.sub.1-C.sub.4 alkyl that
are optionally branched; or R.sup.a and R.sup.b are taken together
with the attached carbon atom to form a carbocyclic ring; R is an
optionally substituted alkyl group, an optionally substituted acyl
group, or a suitably selected nitrogen protecting group; and (*)
indicates points of attachment for the drug, vitamin, imaging
agent, diagnostic agent, other polyvalent linkers, or other parts
of the conjugate.
[0154] In another embodiment, the polyvalent linkers described
herein include compounds of the following formulae
##STR00031##
where m is an integer selected from 1 to about 4; R is an
optionally substituted alkyl group, an optionally substituted acyl
group, or a suitably selected nitrogen protecting group; and (*)
indicates points of attachment for the drug, vitamin, imaging
agent, diagnostic agent, other polyvalent linkers, or other parts
of the conjugate.
[0155] In another embodiment, the polyvalent linkers described
herein include compounds of the following formulae
##STR00032##
where m is an integer selected from 1 to about 4; R is an
optionally substituted alkyl group, an optionally substituted acyl
group, or a suitably selected nitrogen protecting group; and (*)
indicates points of attachment for the drug, vitamin, imaging
agent, diagnostic agent, other polyvalent linkers, or other parts
of the conjugate.
[0156] Another illustrative mechanism involves an arrangement of
the releasable and spacer linkers in such a way that subsequent to
the cleavage of a bond in the polyvalent linker, released
functional groups chemically assist the breakage or cleavage of
additional bonds, also termed anchimeric assisted cleavage or
breakage. An illustrative embodiment of such a polyvalent linker or
portion thereof includes compounds having the formula:
##STR00033##
where X is an heteroatom, such as nitrogen, oxygen, or sulfur, n is
an integer selected from 0, 1, 2, and 3, R is hydrogen, or a
substituent, including a substituent capable of stabilizing a
positive charge inductively or by resonance on the aryl ring, such
as alkoxy, and the like, and either of Z or Z' is the vitamin, or
analog or derivative thereof, or the drug, or analog or derivative
thereof, or a vitamin or drug moiety in conjunction with other
portions of the polyvalent linker. It is appreciated that other
substituents may be present on the aryl ring, the benzyl carbon,
the carbamate nitrogen, the alkanoic acid, or the methylene bridge,
including but not limited to hydroxy, alkyl, alkoxy, alkylthio,
halo, and the like. Assisted cleavage may include mechanisms
involving benzylium intermediates, benzyne intermediates, lactone
cyclization, oxonium intermediates, beta-elimination, and the like.
It is further appreciated that, in addition to fragementation
subsequent to cleavage of the releasable linker, the initial
cleavage of the releasable linker may be facilitated by an
anchimerically assisted mechanism.
[0157] In this embodiment, the hydroxyalkanoic acid, which may
cyclize, facilitates cleavage of the methylene bridge, by for
example an oxonium ion, and facilitates bond cleavage or subsequent
fragmentation after bond cleavage of the releasable linker.
Alternatively, acid catalyzed oxonium ion-assisted cleavage of the
methylene bridge may begin a cascade of fragmentation of this
illustrative polyvalent linker, or fragment thereof. Alternatively,
acid-catalyzed hydrolysis of the carbamate may facilitate the beta
elimination of the hydroxyalkanoic acid, which may cyclize, and
facilitate cleavage of methylene bridge, by for example an oxonium
ion. It is appreciated that other chemical mechanisms of bond
breakage or cleavage under the metabolic, physiological, or
cellular conditions described herein may initiate such a cascade of
fragmentation. It is appreciated that other chemical mechanisms of
bond breakage or cleavage under the metabolic, physiological, or
cellular conditions described herein may initiate such a cascade of
fragmentation.
[0158] Another illustrative embodiment of the linkers described
herein, include releasable linkers that cleave under the conditions
described herein by a chemical mechanism involving beta
elimination. In one aspect, such releasable linkers include
beta-thio, beta-hydroxy, and beta-amino substituted carboxylic
acids and derivatives thereof, such as esters, amides, carbonates,
carbamates, and ureas. In another aspect, such releasable linkers
include 2- and 4-thioarylesters, carbamates, and carbonates.
[0159] In another embodiment, the polyvalent linker includes
additional spacer linkers and releasable linkers connected to form
a polyvalent 3-thiosuccinimid-1-ylalkyloxymethyloxy group,
illustrated by the following formula
##STR00034##
where n is an integer from 1 to 6, the alkyl group is optionally
substituted, and the methyl is optionally substituted with an
additional alkyl or optionally substituted aryl group, each of
which is represented by an independently selected group R. The (*)
symbols indicate points of attachment of the polyvalent linker
fragment to other parts of the conjugates described herein.
[0160] In another embodiment, the polyvalent linker includes
additional spacer linkers and releasable linkers connected to form
a polyvalent 3-thiosuccinimid-1-ylalkylcarbonyl group, illustrated
by the following formula
##STR00035##
where n is an integer from 1 to 6, and the alkyl group is
optionally substituted. The (*) symbols indicate points of
attachment of the polyvalent linker fragment to other parts of the
conjugates described herein. In another embodiment, the polyvalent
linker includes spacer linkers and releasable linkers connected to
form a polyvalent 3-thioalkylsulfonylalkyl(disubstituted silyl)oxy
group, where the disubstituted silyl is substituted with alkyl
and/or optionally substituted aryl groups.
[0161] In another embodiment, the polyvalent linker includes
additional spacer linkers and releasable linkers connected to form
a polyvalent dithioalkylcarbonylhydrazide group, or a polyvalent
3-thiosuccinimid-1-ylalkylcarbonylhydrazide, illustrated by the
following formulae
##STR00036##
where n is an integer from 1 to 6, the alkyl group is optionally
substituted, and the hydrazide form an hydrazone with (B), (D), or
another part of the polyvalent linker (L). The (*) symbols indicate
points of attachment of the polyvalent linker fragment to other
parts of the conjugates described herein.
[0162] In another embodiment, the polyvalent linker includes
additional spacer linkers and releasable linkers connected to form
a polyvalent 3-thiosuccinimid-1-ylalkyloxyalkyloxyalkylidene group,
illustrated by the following formula
##STR00037##
where each n is an independently selected integer from 1 to 6, each
alkyl group independently selected and is optionally substituted,
such as with alkyl or optionally substituted aryl, and where the
alkylidene forms an hydrazone with (B), (D), or another part of the
polyvalent linker (L). The (*) symbols indicate points of
attachment of the polyvalent linker fragment to other parts of the
conjugates described herein.
[0163] Additional illustrative additional spacer linkers include
alkylene-amino-alkylenecarbonyl,
alkylene-thio-carbonylalkylsuccinimid-3-yl, and the like, as
further illustrated by the following formulae:
##STR00038##
where the integers x and y are 1, 2, 3, 4, or 5:
[0164] In another illustrative embodiment, the linker includes one
or more amino acids. In one variation, the linker includes a single
amino acid. In another variation, the linker includes a peptide
having from 2 to about 50, 2 to about 30, or 2 to about 20 amino
acids. In another variation, the linker includes a peptide having
from about 4 to about 8 amino acids. Such amino acids are
illustratively selected from the naturally occurring amino acids,
or stereoisomers thereof. The amino acid may also be any other
amino acid, such as any amino acid having the general formula:
--N(R)--(CR'R'').sub.q--C(O)--
where R is hydrogen, alkyl, acyl, or a suitable nitrogen protecting
group, R' and R'' are hydrogen or a substituent, each of which is
independently selected in each occurrence, and q is an integer such
as 1, 2, 3, 4, or 5. Illustratively, R' and/or R'' independently
correspond to, but are not limited to, hydrogen or the side chains
present on naturally occurring amino acids, such as methyl, benzyl,
hydroxymethyl, thiomethyl, carboxyl, carboxylmethyl,
guanidinopropyl, and the like, and derivatives and protected
derivatives thereof. The above described formula includes all
stereoisomeric variations. For example, the amino acid may be
selected from asparagine, aspartic acid, cysteine, glutamic acid,
lysine, glutamine, arginine, serine, ornitine, threonine, and the
like. In one variation, the releasable linker includes at least 2
amino acids selected from asparagine, aspartic acid, cysteine,
glutamic acid, lysine, glutamine, arginine, serine, ornitine, and
threonine. In another variation, the releasable linker includes
between 2 and about 5 amino acids selected from asparagine,
aspartic acid, cysteine, glutamic acid, lysine, glutamine,
arginine, serine, ornitine, and threonine. In another variation,
the releasable linker includes a tripeptide, tetrapeptide,
pentapeptide, or hexapeptide consisting of amino acids selected
from aspartic acid, cysteine, glutamic acid, lysine, arginine, and
ornitine, and combinations thereof.
[0165] In another illustrative aspect of the vitamin receptor
binding drug delivery conjugate intermediate described herein, the
drug, or an analog or a derivative thereof, includes an alkylthiol
nucleophile.
[0166] Additional linkers are described in the following Tables,
where the (*) atom is the point of attachment of additional spacer
or releaseable linkers, the drug, and/or the binding ligand.
[0167] The following illustrative spacer linkers are described.
TABLE-US-00001 ##STR00039## ##STR00040## ##STR00041## ##STR00042##
##STR00043## ##STR00044## ##STR00045## ##STR00046## ##STR00047##
##STR00048## ##STR00049## ##STR00050## ##STR00051## ##STR00052##
##STR00053## ##STR00054## ##STR00055## ##STR00056## ##STR00057##
##STR00058## ##STR00059## ##STR00060## ##STR00061## ##STR00062##
##STR00063## ##STR00064## ##STR00065## ##STR00066## ##STR00067##
##STR00068## ##STR00069## ##STR00070## ##STR00071## ##STR00072##
##STR00073## ##STR00074## ##STR00075## ##STR00076## ##STR00077##
##STR00078## ##STR00079## ##STR00080## ##STR00081## ##STR00082##
##STR00083## ##STR00084## ##STR00085## ##STR00086##
[0168] The following illustrative releasable linkers are
described.
TABLE-US-00002 ##STR00087## ##STR00088## ##STR00089## ##STR00090##
##STR00091## ##STR00092## ##STR00093## ##STR00094## ##STR00095##
##STR00096## ##STR00097## ##STR00098## ##STR00099## ##STR00100##
##STR00101## ##STR00102## ##STR00103## ##STR00104## ##STR00105##
##STR00106## ##STR00107## ##STR00108## ##STR00109## ##STR00110##
##STR00111## ##STR00112## ##STR00113## ##STR00114## ##STR00115##
##STR00116## ##STR00117## ##STR00118## ##STR00119## ##STR00120##
##STR00121##
[0169] It is appreciated that such hydrophilic linkers may alter
the stability, metabolism and tissue distribution of the
conjugates, especially compared to other conjugate forms such as
the peptidic based forms described in U.S. patent application Ser.
No. 10/765,336. For example, it is understood that in certain
situations, carbohydrate-protein interactions are weaker than
peptide-protein interactions. Thus, it is appreciated that in
various embodiments described herein, the conjugates may lead to
lower binding of serum proteins. These and other physicochemical
differences between the conjugates described herein and others
already reported may include enhanced targeting to target cells and
improved, i.e. more selective or differentially selective
biodistribution profiles. The increased cyctotoxicity may be a
natural consequence of the decreased serum protein binding or the
better or differential biodistribution (i.e. less drug is wasted in
non-specific compartments). This is especially true for the use of
hydrophilic but neutral spacers. Without being bound by theory it
is also suggested that the hydrophilic spacer linkers described
herein may decrease toxicity that might be due at least in part to
non-specific binding interactions.
[0170] In an alternate embodiment, drug is linked to a hydrophilic
spacer linker, directly or indirectly, to accomplish the goal of
decreasing liver clearance. It has been found herein that the
attachment of hydrophilic groups, either releasable or not, and
more specifically hydrophilic neutral groups, increases
renal-specific delivery.
[0171] It has been observed that liver clearance of folate-drug
conjugates possessing disulfide linkers and peptidic spacers retain
residual and sometimes substantial unfavorable toxicity profiles.
Including the hydrophilic spacers described herein also introduced
vectors for kidney-specific delivery. It is therefore appreciated
that including such linkers in targeted drug conjugates may
decrease overall liver uptake and consequentially decrease overall
toxicity. Without being bound by theory, it is appreciated that
toxicity at MTD, such as with vinca alkaloid conjugates, may be
caused by non-specific liver clearance, leading to metabolism,
release of free drug, such as DAVLBH, into bile and then the
intestine. The local toxicity as well as systemic toxicity (due to
re-absorption) might then occur. By including hydrophilic linkers
in the targeted and non-targeted conjugates described herein, it is
believed that clearance through the kidney may preferentially
occur, thus decreasing and/or avoiding concomitant liver metabolism
based toxicity. Accordingly, measuring total bile clearance of the
drug component, such as DAVLBH, from a series of drug-folate
conjugates, may be used to predict which agent would be the least
toxic.
[0172] As described above, the conjugates described herein may be
used to deliver target agents A to cells in a selective or specific
manner. In one aspect of such delivery, unwanted clearance
mechanisms may also be avoided. It has been discovered that the
hydrophilic spacer linkers described herein when used to form
conjugates of receptor binding ligands B and agents A, can decrease
the amount of clearance by the liver. It has further been
discovered that these hydrophilic spacer linkers tend to favor
clearance along renal pathways, such as the kidney. It has further
been discovered that the conjugates described herein exhibit lower
toxicity than the parent agents A by themselves when administered
in the same way. Without being bound by theory, it is suggested
that the lower toxicity arises from the observed decrease in liver
clearance mechanism in favor of renal clearance mechanisms.
[0173] In another embodiment, compounds are described herein that
have reduced uptake by the liver and are less likely to be cleared
by the liver. In one aspect, such compounds are preferentially
cleared by the renal processes as compared to hepatic processes.
Accordingly, in another embodiment, non-targeted compounds of the
following formula are described herein:
L-A
where L is a hydrophilic spacer and A is diagnostic, therapeutic,
or imaging agent. It is appreciated that such non-targeted
compounds, though not targeted using a receptor binding ligand B,
may nonetheless exhibit decreased toxicity than the parent agent A
when delivered in the same manner. The non-targeted compounds, like
the targeted conjugates described above include the hydrophilic
spacer L. Therefore, the agent that does not reach the cell
desirably treated will be cleared by ordinary metabolic and
biological routes. However, it is appreciated that the presence of
the hydrophilic spacer L will direct the clearance through renal
pathways rather than hepatic pathways.
[0174] In another embodiment, multi-drug conjugates are described
herein. Several illustrative configurations of such multi-drug
conjugates are contemplated herein, and include the compounds and
compositions described in PCT international publication No. WO
2007/022494, the disclosure of which is incorporated herein by
reference. Illustratively, the polyvalent linkers may connect the
receptor binding ligand B to the two or more agents A in a variety
of structural configurations, including but not limited to the
following illustrative general formulae:
##STR00122##
where B is the receptor binding ligand, each of (L.sup.1),
(L.sup.2), and (L.sup.3) is a polyvalent linker as described herein
comprising a hydrophilic spacer linker, and optionally including
one or more releasable linkers and/or additional spacer linkers,
and each of (A.sup.1), (A.sup.2), and (A.sup.3) is an agent A, or
an analog or derivative thereof. Other variations, including
additional agents A, or analogs or derivatives thereof, additional
linkers, and additional configurations of the arrangement of each
of (B), (L), and (A), are also contemplated herein.
[0175] In one variation, more than one receptor binding ligand B is
included in the delivery conjugates described herein, including but
not limited to the following illustrative general formulae:
##STR00123##
where each B is a receptor binding ligand, each of (L.sup.1),
(L.sup.2), and (L.sup.3) is a polyvalent linker as described herein
comprising a hydrophilic spacer linker, and optionally including
one or more releasable linkers and/or additional spacer linkers,
and each of (A.sup.1), (A.sup.2), and (A.sup.3) is an agent A, or
an analog or derivative thereof. Other variations, including
additional agents A, or analogs or derivatives thereof, additional
linkers, and additional configurations of the arrangement of each
of (B), (L), and (A), are also contemplated herein. In one
variation, the receptor binding ligands B are ligands for the same
receptor, and in another variation, the receptor binding ligands B
are ligands for different receptors.
[0176] In another illustrative embodiment, the drugs are selected
based on activity against one or more populations of pathogenic
cells with a particular mechanism of action. Illustrative
mechanisms of action include alkylating agents, microtubule
inhibitors, including those that stabilize and/or destabilize
microtubule formation, including beta-tubulin agents, cyclin
dependent kinase (CDK) inhibitors, topoisomerase inhibitors,
protein synthesis inhibitors, protein kinase inhibitors, including
Ras, Raf, PKC, PI3K, and like inhibitors, transcription inhibitor,
antifolates, heat shock protein blockers, and the like.
[0177] Illustrative alkylating agents include, but are not limited
to, mitomycins CBI, and the like. Illustrative cyclin dependent
kinase (CDK) inhibitors include, but are not limited to, CYC202,
seliciclib, R-roscovitine, AGM-1470, and the like. Illustrative
topoisomerase inhibitors include, but are not limited to,
doxorubicin, other anthracyclines, and the like. Illustrative
protein synthesis inhibitors include, but are not limited to,
bruceantin, and the like. Illustrative protein kinase inhibitors,
including Ras, Raf, PKC, PI3K, and like inhibitors, include but are
not limited to L-779,450, R115777, and the like. Illustrative
transcription inhibitors include, but are not limited to,
.alpha.-amanatin, actinomycin, and the like. Illustrative
antifolates include, but are not limited to, methotrexate, and the
like. Illustrative heat shock protein blockers include, but are not
limited to, geldanamycin, and the like.
[0178] Illustrative microtubule inhibitors, including those that
stabilize and/or destabilize microtubule formation, including
.beta.-tubulin agents, microtubule poisons, and the like.
Illustrative microtubule poisons that bind to selected receptors
include, but are not limited to, inhibitors biding to the vinca
binding site such as arenastatin, dolastatin, halichondrin B,
maytansine, phomopsin A, rhizoxin, ustiloxin, vinblastine,
vincristine, and the like, stabilizers binding to the taxol binding
site such as discodermalide, epothilone, taxol, paclitaxol, and the
like, inhibitors binding to the colchicine binding site such as,
colchicine, combretastatin, curacin A, podophyllotoxin,
steganacine, and the like, and others binding to undefined sites
such as cryptophycin, tubulysins, and the like.
[0179] In one embodiment, the tubulsyin is a naturally occurring
tubulysin. In another embodiment, the tubulsyin is a synthetic or
semi-synthetic tubulysin. Additional tubulysin that may be included
in the conjugates described herein are described in PCT
international application serial No. PCT/US2008/056824, the
disclosure of which is incorporated herein by reference.
[0180] In one embodiment of the drug delivery conjugates described
herein, at least one of the drugs is a microtubule inhibitor, or an
analog or derivative thereof. In another embodiment, at least one
of the drugs is a DNA alkylation agent. In another embodiment, at
least one of the drugs is a DNA alkylation agent, and at least one
other of the drugs is a microtubule inhibitor.
[0181] In another embodiment of the drug delivery conjugates
described herein, at least one of the drugs is a P-glycoprotein
(PGP) inhibitor. In another embodiment, at least one of the drugs
included on the drug delivery conjugates described herein is a PGP
inhibitor, and at least one other of the drugs included on the drug
delivery conjugates is a PGP substrate. Illustratively in this
latter embodiment, the PGP substrate is a DNA alkylating agent.
Referring to this embodiment, it is appreciated that pairing a PGP
inhibitor with a PGP substrate, such as a DNA alkylating agent
including, but not limited to, any of the mitomycins like mitomycin
C, mitomycin A, and the like may improve the overall performance of
the drug that is otherwise a PGP substrate. In the releasable
conjugates described herein, the PGP inhibitor drug and the PGP
substrate drug are both released in the cell after endocytosis. In
that manner, the PGP inhibitor drug may improve the overall
efficacy and/or potency of the PGP substrate drug. In addition, the
PGP inhibitor may reduces PGP expression, which in turn will
decrease efflux of one or more of the drugs included on the
multidrug conjugates described herein from the pathogenic cell. It
is appreciated that the mitomycins, or analogs or derivatives
thereof, such as mitomycin C may operate as a PGP inhibitor, or
down-regulator of PGP. It is further appreciated that the vinca
alkaloid, or analog or derivative thereof, such as vinblastine
analogs and derivatives, may be a PGP substrate that is protected
from efflux from the pathogenic cell by the PGP inhibitor or
down-regulator.
[0182] In another embodiment of the drug delivery conjugates
described herein, at least one of the drugs is a vinca alkaloid, or
an analog or derivative thereof. Vinca alklaloids described herein
include all members of the vinca indole-dihydroindole family of
alkaloids, such as but not limited to vindesine, vinblastine,
vincristine, catharanthine, vindoline, leurosine, vinorelbine,
imidocarb, sibutramine, toltrazuril, vinblastinoic acid, and the
like, and analogs and derivatives thereof.
[0183] In another embodiment, methods for treating diseases caused
by or evidenced by pathogenic cell populations are described
herein. The binding ligand (B) drug delivery conjugates can be used
to treat disease states characterized by the presence of a
pathogenic cell population in the host wherein the members of the
pathogenic cell population have an accessible binding site for the
binding ligand (B), or analog or derivative thereof, wherein the
binding site is uniquely expressed, overexpressed, or
preferentially expressed by the pathogenic cells. The selective
elimination of the pathogenic cells is mediated by the binding of
the ligand moiety of the binding ligand (B) drug delivery conjugate
to a ligand receptor, transporter, or other surface-presented
protein that specifically binds the binding ligand (B), or analog
or derivative thereof, and which is uniquely expressed,
overexpressed, or preferentially expressed by the pathogenic cells.
A surface-presented protein uniquely expressed, overexpressed, or
preferentially expressed by the pathogenic cells is a receptor not
present or present at lower concentrations on non-pathogenic cells
providing a means for selective elimination of the pathogenic
cells.
[0184] For example, surface-expressed vitamin receptors, such as
the high-affinity folate receptor, are overexpressed on cancer
cells. Epithelial cancers of the ovary, mammary gland, colon, lung,
nose, throat, and brain have all been reported to express elevated
levels of the folate receptor. In fact, greater than 90% of all
human ovarian tumors are known to express large amounts of this
receptor. Accordingly, the binding ligand (B) drug delivery
conjugates described herein can be used to treat a variety of tumor
cell types, as well as other types of pathogenic cells, such as
infectious agents, that preferentially express ligand receptors,
such as vitamin receptors, and, thus, have surface accessible
binding sites for ligands, such as vitamins, or vitamin analogs or
derivatives. In one aspect, methods are described herein for
targeting binding ligand-linker-drug conjugates to maximize
targeting of the pathogenic cells for elimination.
[0185] The invention further contemplates the use of combinations
of binding ligand-linker-drug conjugates to maximize targeting of
the pathogenic cells for elimination.
[0186] The binding ligand (B) drug delivery conjugates described
herein can be used for both human clinical medicine and veterinary
applications. Thus, the host animal harboring the population of
pathogenic cells and treated with the binding ligand (e.g., a
vitamin) drug delivery conjugates can be human or, in the case of
veterinary applications, can be a laboratory, agricultural,
domestic, or wild animal. The methods described herein can be
applied to host animals including, but not limited to, humans,
laboratory animals such rodents (e.g., mice, rats, hamsters, etc.),
rabbits, monkeys, chimpanzees, domestic animals such as dogs, cats,
and rabbits, agricultural animals such as cows, horses, pigs,
sheep, goats, and wild animals in captivity such as bears, pandas,
lions, tigers, leopards, elephants, zebras, giraffes, gorillas,
dolphins, and whales.
[0187] The methods are applicable to populations of pathogenic
cells that cause a variety of pathologies in these host animals.
The term pathogenic cells refers to for example cancer cells,
infectious agents such as bacteria and viruses, bacteria- or
virus-infected cells, activated macrophages capable of causing a
disease state, and any other type of pathogenic cells that uniquely
express, preferentially express, or overexpress binding ligand
receptors, such as vitamin receptors or receptors that bind analogs
or derivatives of vitamins. Pathogenic cells can also include any
cells causing a disease state for which treatment with the binding
ligand drug delivery conjugates described herein results in
reduction of the symptoms of the disease. For example, the
pathogenic cells can be host cells that are pathogenic under some
circumstances such as cells of the immune system that are
responsible for graft versus host disease, but not pathogenic under
other circumstances.
[0188] Thus, the population of pathogenic cells can be a cancer
cell population that is tumorigenic, including benign tumors and
malignant tumors, or it can be non-tumorigenic. The cancer cell
population can arise spontaneously or by such processes as
mutations present in the germline of the host animal or somatic
mutations, or it can be chemically-, virally-, or
radiation-induced. The methods can be utilized to treat such
cancers as carcinomas, sarcomas, lymphomas, Hodgkin's disease,
melanomas, mesotheliomas, Burkitt's lymphoma, nasopharyngeal
carcinomas, leukemias, and myelomas. The cancer cell population can
include, but is not limited to, oral, thyroid, endocrine, skin,
gastric, esophageal, laryngeal, pancreatic, colon, bladder, bone,
ovarian, cervical, uterine, breast, testicular, prostate, rectal,
kidney, liver, and lung cancers.
[0189] In embodiments where the pathogenic cell population is a
cancer cell population, the effect of conjugate administration is a
therapeutic response measured by reduction or elimination of tumor
mass or of inhibition of tumor cell proliferation. In the case of a
tumor, the elimination can be an elimination of cells of the
primary tumor or of cells that have metastasized or are in the
process of dissociating from the primary tumor. A prophylactic
treatment with the binding ligand (B) drug delivery conjugate
(e.g., a vitamin used as the binding ligand) to prevent return of a
tumor after its removal by any therapeutic approach including
surgical removal of the tumor, radiation therapy, chemotherapy, or
biological therapy is also described. The prophylactic treatment
can be an initial treatment with the binding ligand (B) drug
delivery conjugate, such as treatment in a multiple dose daily
regimen, and/or can be an additional treatment or series of
treatments after an interval of days or months following the
initial treatment(s). Accordingly, elimination of any of the
pathogenic cell populations treated using the described methods
includes reduction in the number of pathogenic cells, inhibition of
proliferation of pathogenic cells, a prophylactic treatment that
prevents return of pathogenic cells, or a treatment of pathogenic
cells that results in reduction of the symptoms of disease.
[0190] In cases where cancer cells are being eliminated, the
methods can be used in combination with surgical removal of a
tumor, radiation therapy, chemotherapy, or biological therapies
such as other immunotherapies including, but not limited to,
monoclonal antibody therapy, treatment with immunomodulatory
agents, adoptive transfer of immune effector cells, treatment with
hematopoietic growth factors, cytokines and vaccination.
[0191] The methods are also applicable to populations of pathogenic
cells that cause a variety of infectious diseases. For example, the
methods are applicable to such populations of pathogenic cells as
bacteria, fungi, including yeasts, viruses, virus-infected cells,
mycoplasma, and parasites. Infectious organisms that can be treated
with the binding ligand (B) drug delivery conjugates described
herein are any art-recognized infectious organisms that cause
pathogenesis in an animal, including such organisms as bacteria
that are gram-negative or gram-positive cocci or bacilli. For
example, Proteus species, Klebsiella species, Providencia species,
Yersinia species, Erwinia species, Enterobacter species, Salmonella
species, Serratia species, Aerobacter species, Escherichia species,
Pseudomonas species, Shigella species, Vibrio species, Aeromonas
species, Campylobacter species, Streptococcus species,
Staphylococcus species, Lactobacillus species, Micrococcus species,
Moraxella species, Bacillus species, Clostridium species,
Corynebacterium species, Eberthella species, Micrococcus species,
Mycobacterium species, Neisseria species, Haemophilus species,
Bacteroides species, Listeria species, Erysipelothrix species,
Acinctobacter species, Brucella species, Pastcurella species,
Vibrio species, Flavobacterium species, Fusobacterium species,
Streptobacillus species, Calymmatobacterium species, Legionella
species, Treponema species, Borrelia species, Leptospira species,
Actinomyces species, Nocardia species, Rickettsia species, and any
other bacterial species that causes disease in a host can be
treated with the binding ligand drug delivery conjugates described
herein.
[0192] Of particular interest are bacteria that are resistant to
antibiotics such as antibiotic-resistant Streptococcus species and
Staphylococcus species, or bacteria that are susceptible to
antibiotics, but cause recurrent infections treated with
antibiotics so that resistant organisms eventually develop.
Bacteria that are susceptible to antibiotics, but cause recurrent
infections treated with antibiotics so that resistant organisms
eventually develop, can be treated with the binding ligand (B) drug
delivery conjugates described herein in the absence of antibiotics,
or in combination with lower doses of antibiotics than would
normally be administered to a patient, to avoid the development of
these antibiotic-resistant bacterial strains.
[0193] Viruses, such as DNA and RNA viruses, can also be treated
with the described methods. Such viruses include, but are not
limited to, DNA viruses such as papilloma viruses, parvoviruses,
adenoviruses, herpesviruses and vaccinia viruses, and RNA viruses,
such as arenaviruses, coronaviruses, rhinoviruses, respiratory
syncytial viruses, influenza viruses, picornaviruses,
paramyxoviruses, reoviruses, retroviruses, lentiviruses, and
rhabdoviruses.
[0194] The methods are also applicable to any fungi, including
yeasts, mycoplasma species, parasites, or other infectious
organisms that cause disease in animals. Examples of fungi that can
be treated with the methods and compositions include fungi that
grow as molds or are yeastlike, including, for example, fungi that
cause diseases such as ringworm, histoplasmosis, blastomycosis,
aspergillosis, cryptococcosis, sporotrichosis, coccidioidomycosis,
paracoccidio-idomycosis, mucormycosis, chromoblastomycosis,
dermatophytosis, protothecosis, fusariosis, pityriasis, mycetoma,
paracoccidioidomycosis, phaeohyphomycosis, pseudallescheriasis,
sporotrichosis, trichosporosis, pneumocystis infection, and
candidiasis.
[0195] The methods can also be utilized to treat parasitic
infections including, but not limited to, infections caused by
tapeworms, such as Taenia, Hymenolepsis, Diphyllobothrium, and
Echinococcus species, flukes, such as Fasciolopsis, Heterophyes,
Metagonimus, Clonorchis, Fasciola, Paragonimus, and Schitosoma
species, roundworms, such as Enterobius, Trichuris, Ascaris,
Ancylostoma, Necator, Strongyloides, Trichinella, Wuchereria,
Brugia, Loa Onchocerca, and Dracunculus species, ameba, such as
Naegleria and Acanthamoeba species, and protozoans, such as
Plasmodium, Trypanosoma, Leishmania, Toxoplasma, Entamoeba,
Giardia, Isospora, Cryptosporidium, and Enterocytozoon species.
[0196] The pathogenic cells to which the binding ligand drug
delivery conjugates described herein are directed can also be cells
harboring endogenous pathogens, such as virus-, mycoplasma-,
parasite-, or bacteria-infected cells, if these cells
preferentially express ligand receptors, such as vitamin
receptors.
[0197] In one embodiment, the binding ligand drug delivery
conjugates can be internalized into the targeted pathogenic cells
upon binding of the binding ligand moiety to a receptor,
transporter, or other surface-presented protein that specifically
binds the ligand and which is preferentially expressed on the
pathogenic cells. Such internalization can occur, for example,
through receptor-mediated endocytosis. If the binding ligand (B)
drug delivery conjugate contains a releasable linker, the binding
ligand moiety and the drug can dissociate intracellularly and the
drug can act on its intracellular target.
[0198] In an alternate embodiment, the binding ligand moiety of the
drug delivery conjugate can bind to the pathogenic cell placing the
drug in close association with the surface of the pathogenic cell.
The drug can then be released by cleavage of the releasable linker.
For example, the drug can be released by a protein disulfide
isomerase if the releasable linker is a disulfide group. The drug
can then be taken up by the pathogenic cell to which the binding
ligand (B) drug delivery conjugate is bound, or the drug can be
taken up by another pathogenic cell in close proximity thereto.
Alternatively, the drug could be released by a protein disulfide
isomerase inside the cell where the releasable linker is a
disulfide group. The drug may also be released by a hydrolytic
mechanism, such as acid-catalyzed hydrolysis, as described above
for certain beta elimination mechanisms, or by an anchimerically
assisted cleavage through an oxonium ion or lactonium ion producing
mechanism. The selection of the releasable linker or linkers will
dictate the mechanism by which the drug is released from the
conjugate. It is appreciated that such a selection can be
pre-defined by the conditions wherein the drug conjugate will be
used. Alternatively, the drug delivery conjugates can be
internalized into the targeted cells upon binding, and the binding
ligand and the drug can remain associated intracellularly with the
drug exhibiting its effects without dissociation from the vitamin
moiety.
[0199] In still another embodiment where the binding ligand is a
vitamin, the vitamin-drug delivery conjugate can act through a
mechanism independent of cellular vitamin receptors. For example,
the drug delivery conjugates can bind to soluble vitamin receptors
present in the serum or to serum proteins, such as albumin,
resulting in prolonged circulation of the conjugates relative to
the unconjugated drug, and in increased activity of the conjugates
towards the pathogenic cell population relative to the unconjugated
drug.
[0200] In another embodiment, where the linker does not comprise a
releasable linker, the vitamin moiety of the drug delivery
conjugate can bind to the pathogenic cell placing the drug on the
surface of the pathogenic cell to target the pathogenic cell for
attack by other molecules capable of binding to the drug.
Alternatively, in this embodiment, the drug delivery conjugates can
be internalized into the targeted cells upon binding, and the
vitamin moiety and the drug can remain associated intracellularly
with the drug exhibiting its effects without dissociation from the
vitamin moiety.
[0201] In another embodiment of this invention, a cell receptor
binding delivery conjugate of the general formula B-L-A is
provided, where L is as defined herein, and A is a drug such as an
immunogen. The immunogen can be a hapten, for example, fluorescein,
dinitrophenyl, and the like. In this embodiment, the vitamin
receptor binding drug delivery conjugate binds to the surface of
the pathogenic cells and "labels" the cells with the immunogen,
thereby triggering an immune response directed at the labeled
pathogenic cell population. Antibodies administered to the host in
a passive immunization or antibodies existing in the host system
from a preexisting innate or acquired immunity bind to the
immunogen and trigger endogenous immune responses. Antibody binding
to the cell-bound vitamin-immunogen conjugate results in
complement-mediated cytotoxicity, antibody-dependent cell-mediated
cytotoxicity, antibody opsonization and phagocytosis,
antibody-induced receptor clustering signaling cell death or
quiescence, or any other humoral or cellular immune response
stimulated by antibody binding to cell-bound ligand-immunogen
conjugates. In cases where an immunogen can be directly recognized
by immune cells without prior antibody opsonization, direct killing
of the pathogenic cells can occur. This embodiment is described in
more detail in U.S. patent application Ser. No. 09/822,379,
incorporated herein by reference. It is appreciated that in certain
variations of this embodiment where the drug is an immunogen, the
polyvalent linker may also include releasable linkers, as described
above, such as a vitamin receptor binding drug delivery conjugate
of the general formula B-L-A where L is a linker that comprises one
or more hydrophilic spacer linkers and a releaseable linker.
[0202] The binding ligand (B) drug delivery conjugates described
herein comprise a binding ligand, a polyvalent linker (L), a drug,
and, optionally, heteroatom linkers to link the binding ligand and
the drug to the polyvalent linker (L). The polyvalent linker (L)
can comprise a spacer linker, a releasable (i.e., cleavable)
linker, and an heteroatom linker, or combinations thereof.
[0203] The drug can be any molecule capable of modulating or
otherwise modifying cell function, including pharmaceutically
active compounds. Suitable molecules can include, but are not
limited to: peptides, oligopeptides, retro-inverso oligopeptides,
proteins, protein analogs in which at least one non-peptide linkage
replaces a peptide linkage, apoproteins, glycoproteins, enzymes,
coenzymes, enzyme inhibitors, amino acids and their derivatives,
receptors and other membrane proteins; antigens and antibodies
thereto; haptens and antibodies thereto; hormones, lipids,
phospholipids, liposomes; toxins; antibiotics; analgesics;
bronchodilators; beta-blockers; antimicrobial agents;
antihypertensive agents; cardiovascular agents including
antiarrhythmics, cardiac glycosides, antianginals and vasodilators;
central nervous system agents including stimulants, psychotropics,
antimanics, and depressants; antiviral agents; antihistamines;
cancer drugs including chemotherapeutic agents; tranquilizers;
anti-depressants; II-2 antagonists; anticonvulsants; antinauseants;
prostaglandins and prostaglandin analogs; muscle relaxants;
anti-inflammatory substances; stimulants; decongestants;
antiemetics; diuretics; antispasmodics; antiasthmatics;
anti-Parkinson agents; expectorants; cough suppressants;
mucolytics; and mineral and nutritional additives.
[0204] Further, the drug can be any drug known in the art which is
cytotoxic, enhances tumor permeability, inhibits tumor cell
proliferation, promotes apoptosis, decreases anti-apoptotic
activity in target cells, is used to treat diseases caused by
infectious agents, enhances an endogenous immune response directed
to the pathogenic cells, or is useful for treating a disease state
caused by any type of pathogenic cell. Drugs suitable for use in
accordance with this invention include adrenocorticoids and
corticosteroids, alkylating agents, antiandrogens, antiestrogens,
androgens, aclamycin and aclamycin derivatives, estrogens,
antimetabolites such as cytosine arabinoside, purine analogs,
pyrimidine analogs, and methotrexate, busulfan, carboplatin,
chlorambucil, cisplatin and other platinum compounds, tamoxiphen,
taxol, paclitaxel, paclitaxel derivatives, Taxotere.RTM.,
cyclophosphamide, daunomycin, daunorubicin, doxorubicin, rhizoxin,
T2 toxin, plant alkaloids, prednisone, hydroxyurea, teniposide,
mitomycins, discodermolides, microtubule inhibitors, epothilones,
tubulysin, cyclopropyl benz[e]indolone, seco-cyclopropyl
benz[e]indolone, O-Ac-seco-cyclopropyl benz[e]indolone, bleomycin
and any other antibiotic, nitrogen mustards, nitrosureas,
vincristine, vinblastine, analogs and derivative thereof such as
deacetylvinblastine monohydrazide, and other vinca alkaloids,
including those described in PCT international publication No. WO
2007/022493, the disclosure of which is incorporated herein by
reference, colchicine, colchicine derivatives, allocolchicine,
thiocolchicine, trityl cysteine, Halicondrin B, dolastatins such as
dolastatin 10, amanitins such as .alpha.-amanitin, camptothecin,
irinotecan, and other camptothecin derivatives thereof,
maytansines, geldanamycin and geldanamycin derivatives,
estramustine, nocodazole, MAP4, colcemid, inflammatory and
proinflammatory agents, peptide and peptidomimetic signal
transduction inhibitors, and any other art-recognized drug or
toxin. Other drugs that can be used in accordance with the
invention include penicillins, cephalosporins, vancomycin,
erythromycin, clindamycin, rifampin, chloramphenicol,
aminoglycoside antibiotics, gentamicin, amphotericin B, acyclovir,
trifluridine, ganciclovir, zidovudine, amantadine, ribavirin, and
any other art-recognized antimicrobial compound.
[0205] In another embodiment, the agent (A) is a drug selected from
a vinca alkaloid, such as DAVLBH, a cryptophycin, bortezomib,
thiobortezomib, a tubulysin, aminopterin, rapamycin, paclitaxel,
docetaxel, doxorubicin, daunorubicin, everolimus, .alpha.-amanatin,
verucarin, didemnin B, geldanomycin, purvalanol A, everolimus,
ispinesib, budesonide, dasatinib, an epothilone, a maytansine, and
a tyrosine kinase inhibitor, including analogs and derivatives of
the foregoing. In another embodiment, the conjugate includes at
least two agents (A) selected from a vinca alkaloid, such as
DAVLBH, a cryptophycin, bortezomib, thiobortezomib, a tubulysin,
aminopterin, rapamycin, paclitaxel, docetaxel, doxorubicin,
daunorubicin, everolimus, .alpha.-amanatin, verucarin, didemnin B,
geldanomycin, purvalanol A, everolimus, ispinesib, budesonide,
dasatinib, an epothilone, a maytansine, and a tyrosine kinase
inhibitor, including analogs and derivatives of the foregoing. In
one variation, the agents (A) are the same. In another variation,
the agents (A) are different.
[0206] In one embodiment, the drugs for use in the methods
described herein remain stable in serum for at least 4 hours. In
another embodiment the drugs have an IC.sub.50 in the nanomolar
range, and, in another embodiment, the drugs are water soluble. If
the drug is not water soluble, the polyvalent linker (L) can be
derivatized to enhance water solubility. The term "drug" also means
any of the drug analogs or derivatives described hereinabove. It
should be appreciated that in accordance with this invention, a
drug analog or derivative can mean a drug that incorporates an
heteroatom through which the drug analog or derivative is
covalently bound to the polyvalent linker (L).
[0207] The binding ligand drug delivery conjugates can comprise a
binding ligand (B), a bivalent linker (L), a drug, and, optionally,
heteroatom linkers to link the binding ligand (B) receptor binding
moiety and the drug to the bivalent linker (L). In one illustrative
embodiment, it should be appreciated that a vitamin analog or
derivative can mean a vitamin that incorporates an heteroatom
through which the vitamin analog or derivative is covalently bound
to the bivalent linker (L). Thus, in this illustrative embodiment,
the vitamin can be covalently bound to the bivalent linker (L)
through an heteroatom linker, or a vitamin analog or derivative
(i.e., incorporating an heteroatom) can be directly bound to the
bivalent linker (L). In similar illustrative embodiments, a drug
analog or derivative is a drug, and a drug analog or derivative can
mean a drug that incorporates an heteroatom through which the drug
analog or derivative is covalently bound to the bivalent linker
(L). Thus, in these illustrative aspects, the drug can be
covalently bound to the bivalent linker (L) through an heteroatom
linker, or a drug analog or derivative (i.e., incorporating an
heteroatom) can be directly bound to the bivalent linker (L). The
bivalent linker (L) can comprise a spacer linker, a releasable
(i.e., cleavable) linker, and an heteroatom linker to link the
spacer linker to the releasable linker in conjugates containing
both of these types of linkers.
[0208] Generally, any manner of forming a conjugate between the
bivalent linker (L) and the binding ligand (B), or analog or
derivative thereof, between the bivalent linker (L) and the drug,
or analog or derivative thereof, including any intervening
heteroatom linkers, can be utilized. Also, any art-recognized
method of forming a conjugate between the spacer linker, the
releasable linker, and the heteroatom linker to form the bivalent
linker (L) can be used. The conjugate can be formed by direct
conjugation of any of these molecules, for example, through
complexation, or through hydrogen, ionic, or covalent bonds.
Covalent bonding can occur, for example, through the formation of
amide, ester, disulfide, or imino bonds between acid, aldehyde,
hydroxy, amino, sulfhydryl, or hydrazo groups.
[0209] In another embodiment, the bivalent linker (L) includes a
chain of atoms selected from C, N, O, S, Si, and P that covalently
connects the binding ligand (B), the hydrophilic linker, and/or the
agent (A). The linker may have a wide variety of lengths, such as
in the range from about 2 to about 100 atoms. The atoms used in
forming the linker may be combined in all chemically relevant ways,
such as chains of carbon atoms forming alkylene, alkenylene, and
alkynylene groups, and the like; chains of carbon and oxygen atoms
forming ethers, polyoxyalkylene groups, or when combined with
carbonyl groups forming esters and carbonates, and the like; chains
of carbon and nitrogen atoms forming amines, imines, polyamines,
hydrazines, hydrazones, or when combined with carbonyl groups
forming amides, ureas, semicarbazides, carbazides, and the like;
chains of carbon, nitrogen, and oxygen atoms forming alkoxyamines,
alkoxylamines, or when combined with carbonyl groups forming
urethanes, amino acids, acyloxylamines, hydroxamic acids, and the
like; and many others. In addition, it is to be understood that the
atoms forming the chain in each of the foregoing illustrative
embodiments may be either saturated or unsaturated, such that for
example, alkanes, alkenes, alkynes, imines, and the like may be
radicals that are included in the linker. In addition, it is to be
understood that the atoms forming the linker may also be cyclized
upon each other to form divalent cyclic structures that form the
linker, including cyclo alkanes, cyclic ethers, cyclic amines,
arylenes, heteroarylenes, and the like in the linker.
[0210] In another embodiment, pharmaceutical compositions
comprising an amount of a binding ligand (B) drug delivery
conjugate effective to eliminate a population of pathogenic cells
in a host animal when administered in one or more doses are
described. The binding ligand drug delivery conjugate is preferably
administered to the host animal parenterally, e.g., intradermally,
subcutaneously, intramuscularly, intraperitoneally, intravenously,
or intrathecally. Alternatively, the binding ligand drug delivery
conjugate can be administered to the host animal by other medically
useful processes, such as orally, and any effective dose and
suitable therapeutic dosage form, including prolonged release
dosage forms, can be used.
[0211] Examples of parenteral dosage forms include aqueous
solutions of the active agent, in an isotonic saline, 5% glucose or
other well-known pharmaceutically acceptable liquid carriers such
as liquid alcohols, glycols, esters, and amides. The parenteral
dosage form can be in the form of a reconstitutable lyophilizate
comprising the dose of the drug delivery conjugate. In one aspect
of the present embodiment, any of a number of prolonged release
dosage forms known in the art can be administered such as, for
example, the biodegradable carbohydrate matrices described in U.S.
Pat. Nos. 4,713,249; 5,266,333; and 5,417,982, the disclosures of
which are incorporated herein by reference, or, alternatively, a
slow pump (e.g., an osmotic pump) can be used.
[0212] In one illustrative aspect, at least one additional
composition comprising a therapeutic factor can be administered to
the host in combination or as an adjuvant to the above-detailed
methodology, to enhance the binding ligand drug delivery
conjugate-mediated elimination of the population of pathogenic
cells, or more than one additional therapeutic factor can be
administered. The therapeutic factor can be selected from a
chemotherapeutic agent, or another therapeutic factor capable of
complementing the efficacy of the administered binding ligand drug
delivery conjugate.
[0213] In one illustrative aspect, therapeutically effective
combinations of these factors can be used. In one embodiment, for
example, therapeutically effective amounts of the therapeutic
factor, for example, in amounts ranging from about 0.1
MIU/m.sup.2/dose/day to about 15 MIU/m.sup.2/dose/day in a multiple
dose daily regimen, or for example, in amounts ranging from about
0.1 MIU/m.sup.2/dose/day to about 7.5 MIU/m.sup.2/dose/day in a
multiple dose daily regimen, can be used along with the binding
ligand drug delivery conjugates to eliminate, reduce, or neutralize
pathogenic cells in a host animal harboring the pathogenic cells
(MIU=million international units; m.sup.2=approximate body surface
area of an average human).
[0214] In another embodiment, chemotherapeutic agents, which are,
for example, cytotoxic themselves or can work to enhance tumor
permeability, are also suitable for use in the described methods in
combination with the binding ligand drug delivery conjugates. Such
chemotherapeutic agents include adrenocorticoids and
corticosteroids, alkylating agents, antiandrogens, antiestrogens,
androgens, aclamycin and aclamycin derivatives, estrogens,
antimetabolites such as cytosine arabinoside, purine analogs,
pyrimidine analogs, and methotrexate, busulfan, carboplatin,
chlorambucil, cisplatin and other platinum compounds, tamoxiphen,
taxol, paclitaxel, paclitaxel derivatives, Taxotere.RTM.,
cyclophosphamide, daunomycin, daunorubicin, doxorubicin, rhizoxin,
T2 toxin, plant alkaloids, prednisone, hydroxyurea, teniposide,
mitomycins, discodermolides, microtubule inhibitors, epothilones,
tubulysin, cyclopropyl benz[e]indolone, seco-cyclopropyl
benz[e]indolone, O-Ac-seco-cyclopropyl benz[e]indolone, bleomycin
and any other antibiotic, nitrogen mustards, nitrosureas,
vincristine, vinblastine, and analogs and derivative thereof such
as deacetylvinblastine monohydrazide, colchicine, colchicine
derivatives, allocolchicine, thiocolchicine, trityl cysteine,
IIalicondrin B, dolastatins such as dolastatin 10, amanitins such
as .alpha.-amanitin, camptothecin, irinotecan, and other
camptothecin derivatives thereof, geldanamycin and geldanamycin
derivatives, estramustine, nocodazole, MAP4, colcemid, inflammatory
and proinflammatory agents, peptide and peptidomimetic signal
transduction inhibitors, and any other art-recognized drug or
toxin. Other drugs that can be used include penicillins,
cephalosporins, vancomycin, erythromycin, clindamycin, rifampin,
chloramphenicol, aminoglycoside antibiotics, gentamicin,
amphotericin B, acyclovir, trifluridine, ganciclovir, zidovudine,
amantadine, ribavirin, maytansines and analogs and derivatives
thereof, gemcitabine, and any other art-recognized antimicrobial
compound.
[0215] The therapeutic factor can be administered to the host
animal prior to, after, or at the same time as the binding ligand
drug delivery conjugates and the therapeutic factor can be
administered as part of the same composition containing the binding
ligand drug delivery conjugate or as part of a different
composition than the binding ligand drug delivery conjugate. Any
such therapeutic composition containing the therapeutic factor at a
therapeutically effective dose can be used.
[0216] Additionally, more than one type of binding ligand drug
delivery conjugate can be used. Illustratively, for example, the
host animal can be treated with conjugates with different vitamins,
but the same drug in a co-dosing protocol. In other embodiments,
the host animal can be treated with conjugates comprising the same
binding ligand linked to different drugs, or various binding
ligands linked to various drugs. In another illustrative
embodiment, binding ligand drug delivery conjugates with the same
or different vitamins, and the same or different drugs comprising
multiple vitamins and multiple drugs as part of the same drug
delivery conjugate could be used.
[0217] The unitary daily dosage of the binding ligand drug delivery
conjugate can vary significantly depending on the host condition,
the disease state being treated, the molecular weight of the
conjugate, its route of administration and tissue distribution, and
the possibility of co-usage of other therapeutic treatments such as
radiation therapy. The effective amount to be administered to a
patient is based on body surface area, patient weight, and
physician assessment of patient condition. In illustrative
embodiments, effective doses can range, for example, from about 1
ng/kg to about 1 mg/kg, from about 1 .mu.g/kg to about 500
.mu.g/kg, from about 1 .mu.g/kg to about 100 .mu.g/kg, from about 1
.mu.g/kg to about 50 .mu.g/kg, and from about 1 .mu.g/kg to about
10 .mu.g/kg.
[0218] In another illustrative aspect, any effective regimen for
administering the binding ligand drug delivery conjugates can be
used. For example, the binding ligand drug delivery conjugates can
be administered as single doses, or can be divided and administered
as a multiple-dose daily regimen. In other embodiments, a staggered
regimen, for example, one to three days per week can be used as an
alternative to daily treatment, and such intermittent or staggered
daily regimen is considered to be equivalent to every day treatment
and within the scope of the methods described herein. In one
embodiment, the host is treated with multiple injections of the
binding ligand drug delivery conjugate to eliminate the population
of pathogenic cells. In another embodiment, the host is injected
multiple times (preferably about 2 up to about 50 times) with the
binding ligand drug delivery conjugate, for example, at 12-72 hour
intervals or at 48-72 hour intervals. In other embodiments,
additional injections of the binding ligand drug delivery conjugate
can be administered to the patient at an interval of days or months
after the initial injections(s) and the additional injections
prevent recurrence of the disease state caused by the pathogenic
cells.
[0219] In one embodiment, vitamins, or analogs or derivatives
thereof, that can be used in the binding ligand drug delivery
conjugates include those that bind to receptors expressed
specifically on activated macrophages, such as the folate receptor
which binds folate, or an analog or derivative thereof. The
folate-linked conjugates, for example, can be used to kill or
suppress the activity of activated macrophages that cause disease
states in the host. Such macrophage targeting conjugates, when
administered to a patient suffering from an activated
macrophage-mediated disease state, work to concentrate and
associate the conjugated drug in the population of activated
macrophages to kill the activated macrophages or suppress
macrophage function. Elimination, reduction, or deactivation of the
activated macrophage population works to stop or reduce the
activated macrophage-mediated pathogenesis characteristic of the
disease state being treated. Exemplary of diseases known to be
mediated by activated macrophages include rheumatoid arthritis,
ulcerative colitis, Crohn's disease, psoriasis, osteomyelitis,
multiple sclerosis, atherosclerosis, pulmonary fibrosis,
sarcoidosis, systemic sclerosis, organ transplant rejection (GVHD)
and chronic inflammations. Administration of the drug delivery
conjugate is typically continued until symptoms of the disease
state are reduced or eliminated.
[0220] Illustratively, the binding ligand drug delivery conjugates
administered to kill activated macrophages or suppress the function
of activated macrophages can be administered parenterally to the
animal or patient suffering from the disease state, for example,
intradermally, subcutaneously, intramuscularly, intraperitoneally,
or intravenously in combination with a pharmaceutically acceptable
carrier. In another embodiment, the binding ligand drug delivery
conjugates can be administered to the animal or patient by other
medically useful procedures and effective doses can be administered
in standard or prolonged release dosage forms. In another aspect,
the therapeutic method can be used alone or in combination with
other therapeutic methods recognized for treatment of disease
states mediated by activated macrophages.
[0221] The drug delivery conjugates described herein can be
prepared by art-recognized synthetic methods. The synthetic methods
are chosen depending upon the selection of the optionally addition
heteroatoms or the heteroatoms that are already present on the
spacer linkers, releasable linkers, the drug, and/or or the binding
ligand. In general, the relevant bond forming reactions are
described in Richard C. Larock, "Comprehensive Organic
Transformations, a guide to functional group preparations," VCH
Publishers, Inc. New York (1989), and in Theodora E. Greene &
Peter G. M. Wuts, "Protective Groups ion Organic Synthesis," 2d
edition, John Wiley & Sons, Inc. New York (1991), the
disclosures of which are incorporated herein by reference.
Additional details for preparing functional groups, including
amides and esters, ketals and acetals, succinimides, silyloxys,
hydrazones, acyl hydrazines, semicarbazones, disulfides,
carbonates, sulfonates, and the like contained in the linker,
including releasable linkers are described in U.S. patent
application publication No. US 2005/0002942 A1, incorporated herein
in its entirety by reference.
[0222] General formation of folate-peptides. The folate-containing
peptidyl fragment Pte-Glu-(AA).sub.n-NH(CHR.sub.2)CO.sub.2H (3) is
prepared by a polymer-supported sequential approach using standard
methods, such as the Fmoc-strategy on an acid-sensitive
Fmoc-AA-Wang resin (1), as shown in Scheme 1.
##STR00124##
[0223] In this illustrative embodiment of the processes described
herein, R.sub.1 is Fmoc, R.sub.2 is the desired
appropriately-protected amino acid side chain, and DIPEA is
diisopropylethylamine. Standard coupling procedures, such as PyBOP
and others described herein or known in the art are used, where the
coupling agent is illustratively applied as the activating reagent
to ensure efficient coupling. Fmoc protecting groups are removed
after each coupling step under standard conditions, such as upon
treatment with piperidine, tetrabutylammonium fluoride (TBAF), and
the like. Appropriately protected amino acid building blocks, such
as Fmoc-Glu-OtBu, N.sup.10-TFA-Pte-OH, and the like, are used, as
described in Scheme 1, and represented in step (b) by Fmoc-AA-OH.
Thus, AA refers to any amino acid starting material, that is
appropriatedly protected. It is to be understood that the term
amino acid as used herein is intended to refer to any reagent
having both an amine and a carboxylic acid functional group
separated by one or more carbons, and includes the naturally
occurring alpha and beta amino acids, as well as amino acid
derivatives and analogs of these amino acids. In particular, amino
acids having side chains that are protected, such as protected
serine, threonine, cysteine, aspartate, and the like may also be
used in the folate-peptide synthesis described herein. Further,
gamma, delta, or longer homologous amino acids may also be included
as starting materials in the folate-peptide synthesis described
herein. Further, amino acid analogs having homologous side chains,
or alternate branching structures, such as norleucine, isovaline,
.beta.-methyl threonine, .beta.-methyl cysteine,
.beta.,.beta.-dimethyl cysteine, and the like, may also be included
as starting materials in the folate-peptide synthesis described
herein.
[0224] The coupling sequence (steps (a) & (b)) involving
Fmoc-AA-OH is performed "n" times to prepare solid-support peptide
2, where n is an integer and may equal 0 to about 100. Following
the last coupling step, the remaining Fmoc group is removed (step
(a)), and the peptide is sequentially coupled to a glutamate
derivative (step (c)), deprotected, and coupled to TFA-protected
pteroic acid (step (d)). Subsequently, the peptide is cleaved from
the polymeric support upon treatment with trifluoroacetic acid,
ethanedithiol, and triisopropylsilane (step (e)). These reaction
conditions result in the simultaneous removal of the t-Bu, t-Boc,
and Trt protecting groups that may form part of the
appropriately-protected amino acid side chain. The TFA protecting
group is removed upon treatment with base (step (f)) to provide the
folate-containing peptidyl fragment 3.
[0225] In addition, the following illustrative process may be used
to prepare compounds described herein, where is an integer such as
1 to about 10.
##STR00125##
It is to be understood that although the foregoing synthetic
process is illustrated for selected compounds, such as the
particular saccharopeptides shown, additional analogous compounds
may be prepared using the same or similar process by the routine
selection of starting materials and the routine optimization of
reaction conditions.
[0226] The compounds described herein may be prepared using
conventional synthetic organic chemistry. In addition, the
following illustrative process may be used to prepare compounds
described herein, where is an integer such as 1 to about 10.
##STR00126##
It is to be understood that although the foregoing synthetic
process is illustrated for selected compounds, such as the
particular saccharopeptides shown, additional analogous compounds
may be prepared using the same or similar process by the routine
selection of starting materials and the routine optimization of
reaction conditions.
[0227] In addition, the following illustrative process may be used
to prepare compounds described herein.
##STR00127##
It is to be understood that although the foregoing synthetic
process is illustrated for selected compounds, additional analogous
compounds may be prepared using the same or similar process by the
routine selection of starting materials and the routine
optimization of reaction conditions.
[0228] In each of the foregoing synthetic processes, the
intermediates may be coupled with any additional hydrophilic spacer
linkers, other spacer linkers, releasable linkers, or the agent A.
In variations of each of the foregoing processes, additional
hydrophilic spacer linkers, other spacer linkers, or releasable
linkers may be inserted between the binding ligand B and the
indicated hydrophilic spacer linkers. In addition, it is to be
understood that the left-to-right arrangement of the bivalent
hydrophilic spacer linkers is not limiting, and accordingly, the
agent A, the binding ligand B, additional hydrophilic spacer
linkers, other spacer linkers, and/or releasable linkers may be
attached to either end of the hydrophilic spacer linkers described
herein.
Method Examples
[0229] Relative Affinity Assay. The affinity for folate receptors
(FRs) relative to folate was determined according to a previously
described method (Westerhof, G. R., J. H. Schornaget, et al. (1995)
Mol. Pharm. 48: 459-471) with slight modification. Briefly,
FR-positive KB cells were heavily seeded into 24-well cell culture
plates and allowed to adhere to the plastic for 18 h. Spent
incubation media was replaced in designated wells with folate-free
RPMI (FFRPMI) supplemented with 100 nM 3H-folic acid in the absence
and presence of increasing concentrations of test article or folic
acid. Cells were incubated for 60 min at 37.degree. C. and then
rinsed 3 times with PBS, pH 7.4. Five hundred microliters of 1% SDS
in PBS, pH 7.4, were added per well. Cell lysates were then
collected and added to individual vials containing 5 mL of
scintillation cocktail, and then counted for radioactivity.
Negative control tubes contained only the .sup.3H-folic acid in
FFRPMI (no competitor). Positive control tubes contained a final
concentration of 1 mM folic acid, and CPMs measured in these
samples (representing non-specific binding of label) were
subtracted from all samples. Notably, relative affinities were
defined as the inverse motar ratio of compound required to displace
50% of .sup.3H-folic acid bound to the FR on KB cells, and the
relative affinity of folic acid for the FR was set to 1.
[0230] Inhibition of Cellular DNA Synthesis. The compounds
described herein were evaluated using an in vitro cytotoxicity
assay that predicts the ability of the drug to inhibit the growth
of folate receptor-positive KB cells. The compounds were comprised
of folate linked to a respective chemotherapeutic drug, as prepared
according to the protocols described herein. The KB cells were
exposed for up to 7 h at 37.degree. C. to the indicated
concentrations of folate-drug conjugate in the absence or presence
of at least a 100-fold excess of folic acid. The cells were then
rinsed once with fresh culture medium and incubated in fresh
culture medium for 72 hours at 37.degree. C. Cell viability was
assessed using a .sup.3H-thymidine incorporation assay.
[0231] In Vitro Concentration-Dependent Cytotoxic Activity. Cells
were heavily seeded in 24-well Falcon plates and allowed to form
nearly confluent monolayers overnight. Thirty minutes prior to the
addition of test article, spent medium was aspirated from all wells
and replaced with fresh folate-free RPMI (FFRPMI). Note, designated
wells received media containing 100 .mu.M folic acid; and, cells
within the latter wells were used to determine the targeting
specificity, since cytotoxic activity produced in the presence of
excess folic acid (enables competition for FR binding) would
signify the portion of the total activity that was unrelated to
FR-specific delivery. Following one rinse with 1 mL of fresh FFRPMI
containing 10% heat-inactivated fetal calf serum, each well
received 1 mL of media containing increasing concentrations of test
article (4 wells per sample) in the presence or absence of 100 M
free folic acid (a binding site competitor). Treated cells were
pulsed for 2 h at 37.degree. C., rinsed 4 times with 0.5 mL of
media, and then chased in 1 mL of fresh media up to 70 h. Spent
media was aspirated from all wells and replaced with fresh media
containing 5 .mu.Ci/mL 3H-thymidine. Following a further 2 h
37.degree. C. incubation, cells were washed 3 times with 0.5 mL of
PBS and then treated with 0.5 mL of ice-cold 5% trichloroacetic
acid per well. After 15 min, the trichloroacetic acid was aspirated
and the cell material solubilized by the addition of 0.5 mL of 0.25
N sodium hydroxide for 15 min. Four hundred and fifty .mu.L of each
solubilized sample were transferred to scintillation vials
containing 3 mL of Ecolume scintillation cocktail and then counted
in a liquid scintillation counter. Final tabulated results were
expressed as the percentage of .sup.3H-thymidine incorporation
relative to untreated controls.
[0232] As shown in the figures herein, dose-dependent cytotoxicity
was measurable, and in most cases, the IC.sub.50 values
(concentration of drug conjugate required to reduce
.sup.3H-thymidine incorporation into newly synthesized DNA by 50%)
were in the low nanomolar range. Furthermore, the cytotoxicities of
these conjugates were reduced in the presence of excess free folic
acid, indicating that the observed cell killing was mediated by
binding to the folate receptor. The following table illustrated
data for seleted compounds against KB cells and against RAW264.7
cells
TABLE-US-00003 KB Cells RAW264.7 Cells Conjugate IC.sub.50
Competable IC.sub.50 Competable Number Base Drug(s) (nM) with xs
folate (nM) with xs folate EC0234 DAVLBH 56 Yes EC0246 DAVLBH Yes
EC0258 DAVLBH 8.4 Yes EC0262 cryptophycin 4 Yes EC0263 DAVLBH 11
Yes EC0409 DAVLBH 7 Yes EC0525 Thio-bortezomib 68 Yes EC0543
Tubulysin A 1.6 Yes EC0551 Aminopterin 1 Yes EC0552 Rapamycin 100
Yes EC0561 Paclitaxel 53 Yes EC0563 Thiobortezomib + 387 Yes
Rapamycin EC0582 Thio-bortezomib + 51 Yes Everolimus EC0592
.alpha.-amanatin -3 Yes 5 Yes EC0595 Bis-Thio- 4 Yes bortezomib
EC0598 Verucarin 33 Yes EC0605 Bis-Verucarin 14 Yes EC0610 Didemnin
B 4 Yes EC0647 Bis-Aminopterin 0.3 Yes
[0233] In Vitro Test against Various Cancer Cell Lines. Cells are
heavily seeded in 24-well Falcon plates and allowed to form nearly
confluent monolayers overnight. Thirty minutes prior to the
addition of the test compound, spent medium is aspirated from all
wells and replaced with fresh folate-deficient RPMI medium
(FFRPMI). A subset of wells are designated to receive media
containing 100 M folic acid. The cells in the designated wells are
used to determine the targeting specificity. Without being bound by
theory it is suggested that the cytotoxic activity produced by test
compounds in the presence of excess folic acid, i.e. where there is
competition for FR binding, corresponds to the portion of the total
activity that is unrelated to FR-specific delivery. Following one
rinse with 1 mL of fresh FFRPMI containing 10% heat-inactivated
fetal calf serum, each well receives 1 mL of medium containing
increasing concentrations of test compound (4 wells per sample) in
the presence or absence of 100 M free folic acid as indicated.
Treated cells are pulsed for 2 h at 37.degree. C., rinsed 4 times
with 0.5 mL of media, and then chased in 1 mL of fresh medium up to
70 h. Spent medium is aspirated from all wells and replaced with
fresh medium containing 5 .mu.Ci/mL .sup.3H-thymidine. Following a
further 2 h 37.degree. C. incubation, cells are washed 3 times with
0.5 mL of PBS and then treated with 0.5 mL of ice-cold 5%
trichloroacetic acid per well. After 15 min, the trichloroacetic
acid is aspirated and the cell material solubilized by the addition
of 0.5 mL of 0.25 N sodium hydroxide for 15 min. A 450 .mu.L
aliquot of each solubilized sample is transferred to a
scintillation vial containing 3 mL of Ecolume scintillation
cocktail and then counted in a liquid scintillation counter. Final
tabulated results are expressed as the percentage of
.sup.3H-thymidine incorporation relative to untreated controls.
[0234] Inhibition of Tumor Growth in Mice. Four to seven week-old
mice (Balb/c or nu/nu strains) were purchased from Harlan Sprague
Dawley, Inc. (Indianapolis, Ind.). Normal rodent chow contains a
high concentration of folic acid (6 mg/kg chow); accordingly, mice
used were maintained on the folate-free diet (Harlan diet #TD00434)
for 1 week before tumor implantation to achieve serum folate
concentrations close to the range of normal human serum. For tumor
cell inoculation, 1.times.10.sup.6 M109 cells (Balb/c strain) or
1.times.10.sup.6 KB cells (nu/nu strain) in 100 .mu.L were injected
in the subcutis of the dorsal medial area. Tumors were measured in
two perpendicular directions every 2-3 days using a caliper, and
their volumes were calculated as 0.5.times.L.times.W.sup.2, where
L=measurement of longest axis in mm and W=measurement of axis
perpendicular to L in mm. Log cell kill (LCK) and treated over
control (T/C) values were then calculated according to published
procedures (see, e.g., Lee et al., "BMS-247550: a novel epothilone
analog with a mode of action similar to paclitaxel but possessing
superior antitumor efficacy" Clin Cancer Res 7:1429-1437 (2001);
Rose, "Taxol-based combination chemotherapy and other in vivo
preclinical antitumor studies" J Natl Cancer Inst Monogr 47-53
(1993)). Dosing solutions were prepared fresh each day in PBS and
administered through the lateral tail vein of the mice. Dosing was
initiated when the s.c. tumors had an average volume between 50-100
mm.sup.3 (t.sub.0), typically 8 days post tumor inoculation (PTI)
for KB tumors, and 11 days PTI for M109 tumors.
[0235] General KB Tumor Assay. The anti-tumor activity of the
compounds described herein, when administered intravenously (i.v.)
to tumor-bearing animals, was evaluated in nu/nu mice bearing
subcutaneous KB tumors. Approximately 8 days post tumor inoculation
in the subcutis of the right axilla with 1.times.10.sup.6 KB cells
(average tumor volume at t.sub.0=50-100 mm.sup.3), in mice
(5/group) were injected i.v. three times a week (T1W), for 3 weeks
with 5 mol/kg of the drug delivery conjugate or with an equivalent
dose volume of PBS (control), unless otherwise indicated. Tumor
growth was measured using calipers at 2-day or 3-day intervals in
each treatment group. Tumor volumes were calculated using the
equation V=a.times.b.sup.2/2, where "a" is the length of the tumor
and "b" is the width expressed in millimeters.
[0236] General M109 Tumors Assay. The anti-tumor activity of the
compounds described herein, when administered intravenously (i.v.)
to tumor-bearing animals, was evaluated in Balb/c mice bearing
subcutaneous M109 tumors (a syngeneic lung carcinoma).
Approximately 11 days post tumor inoculation in the subcutis of the
right axilla with 1.times.10.sup.6 M109 cells (average tumor volume
at t.sub.0=60 mm.sup.3), mice (5/group) were injected i.v. three
times a week (TIW), for 3 weeks with 1500 nmol/kg of the drug
delivery conjugate or with an equivalent dose volume of PBS
(control). Tumor growth was measured using calipers at 2-day or
3-day intervals in each treatment group. Tumor volumes were
calculated using the equation V=a.times.b.sup.2/2, where "a" is the
length of the tumor and "b" is the width expressed in
millimeters.
[0237] General 4T-1 Tumor Assay. Six to seven week-old mice (female
Balb/c strain) were obtained from Harlan, Inc., Indianapolis, Ind.
The mice were maintained on Harlan's folate-free chow for a total
of three weeks prior to the onset of and during this experiment.
Folate receptor-negative 4T-1 tumor cells (1.times.10.sup.6 cells
per animal) were inoculated in the subcutis of the right axilla.
Approximately 5 days post tumor inoculation when the 4T-1 tumor
average volume was .about.100 mm.sup.3, mice (5/group) were
injected i.v. three times a week (TIW), for 3 weeks with 3 mol/kg
of drug delivery conjugate or with an equivalent dose volume of PBS
(control), unless otherwise indicated herein. Tumor growth was
measured using calipers at 2-day or 3-day intervals in each
treatment group. Tumor volumes were calculated using the equation
V=a.times.b.sup.2/2, where "a" is the length of the tumor and "b"
is the width expressed in millimeters.
[0238] The data shown in FIGS. 3A, 4A, 5A, 6A, 7A, 8A, and 10A
indicate that the conjugates described herein exhibit superior
efficacy in the treatment of tumors compared to the corresponding
unconjugated compounds. Treatment of Balb/c mice with s.c. M109
tumors with EC0396 and EC145 (FIG. 4A) led to complete responses in
all treated animals (3/3 for EC0396 and 5/5 for EC145). In
addition, after nearly 70 days, no recurrence of disease was
observed. Similarly, treatment with EC0400 (FIG. 5A) led to a
complete response and no recurrence of disease after nearly 70
days. Treatment with the conjugated compounds described herein
including a hydrophilic spacer linker (e.g. EC0436) were superior
to comparison conjugates lacking hydrophilic spacer linkers (e.g.
EC0305) showed superior efficacy (FIG. 8A). EC0436 showed a
complete response in 5/5 animals with no recurrence of disease
after 90 days.
[0239] Drug Toxicity Determinations. Persistent drug toxicity was
assessed by collecting blood via cardiac puncture and submitting
the serum for independent analysis of blood urea nitrogen (BUN),
creatinine, total protein, AST-SGOT, ALT-SGPT plus a standard
hematological cell panel at Ani-Lytics, Inc. (Gaithersburg, Md.).
In addition, histopathologic evaluation of formalin-fixed heart,
lungs, liver, spleen, kidney, intestine, skeletal muscle and bone
(tibia/fibula) were conducted by board-certified pathologists at
Animal Reference Pathology Laboratories (ARUP; Salt Lake City,
Utah).
[0240] Toxicity as Measured by Weight Loss. The percentage weight
change was determined in mice (5 mice/group) on selected days
post-tumor inoculation (PTI), compared to controls, and graphed. As
shown in FIGS. 3B, 4B, 5B, 6B, 7B, 8B, and 10B, the conjugated
compounds described herein showed equal or less toxicity compared
to unconjugated compounds, as determined by percent weight
loss.
[0241] Single and Multiple Dose MTD.sub.app on Mice. The compounds
described herein may show a positive relationship between the
number of hydrophilic spacer linkers included in the conjugate and
the maximum tolerated dose on mice for a single dose. For example
the following vinblastine conjugates described herein compared to a
control conjugate are shown in the following table.
TABLE-US-00004 No. of hydrophilic Single Dose MTD.sub.app Compound
linkers (.mu.mol/kg) EC145 0 15 EC0234 1 12* EC0246 2 <20**
EC0263 3 >20 *dose limited by solubility; **1/3 mice died at 20
.mu.mol/kg.
EC0436 and Comparative Example EC0305 were also administered i.v.
to Balb/c mice TIW for 1 week. The resulting MTD for the multiple
dose was EC0305 (6 mmol/kg) and EC0436 (9 mmol/kg). The data
indicate that EC0436 can be dosed at levels 50% greater than
EC0305.
[0242] Serum Binding. Serum binding of Folate-DAVLBH conjugates
containing hydrophilic spacer linkers compared to Comparative
Example EC145 lacking a hydrophilic spacer linker 50 .mu.M compound
in serum with 30K NMWL filtration and evaluation by HPLC-UV
detection (n=3).
TABLE-US-00005 Human Serum Mouse Serum Compound (% Bound) SD (%
Bound) SD EC145 54.3 1.6 67.3 2.6 EC0396 42.7 4.4 72.2 5.2 EC0400
61.1 1.9 75.5 1.4
[0243] Bile Clearance. Comparison of Bile Clearance (% ID) of
unconjugated drug, drug conjugate lacking a hydrophilic spacer
linker, and conjugates described herein.
TABLE-US-00006 Bile clearance Compound Spacer (% ID) DAVLBH None
58.0 EC145 no hydrophilic spacer 8.7 EC0234 Mono-ribosyl 10.6
EC0246 Bis-ribosyl 4.7 EC0258 Tri-ribosyl 3.2 EC0434 Tetra-ribosyl
2.8 EC0400 Mono-glucuronide 6.3 EC0423 Bis-glucuronide 3.9 EC0409
PEG.sub.12 7.9 EC0429 Piperazine/Asp 8.6
[0244] The results shown in FIGS. 11 and 13 indicate a 76% decrease
in the liver clearance of EC0434, which includes hydrophilic spacer
linkers described herein, as compared to the standard EC145.
Without being bound by theory, these results are believed to
correspond to non-specific liver clearance, and accordingly, it is
suggested that significantly lower doses of those conjugates that
include the hydrophilic spacer linkers described herein may be
administered compared to the corresponding conjugates that do not
include such linkers. Further, without being bound by theory, it is
suggested that hepatic clearance leads to the dose limiting
GI-related toxicity that is observed with some conjugates.
[0245] Western Blot Analysis. The data shown in FIG. 13 indicate
that EC0565 (folate-sugar-everolimus) can cause a dose-dependent,
and specific knockdown of the downstream targets of mTOR
(intracellular target for everolimus). Without being bound by
theory, in it believed that folate delivers everolimus inside the
cell where everolimus inhibits mTOR, which is the mammalian target
of rapamycin and a ser/thr kinase. Inhibition of mTOR's downstream
targets (P70 S6-kinase and Ribosomal S6) results, as shown on the
Western blot.
Compound Examples
##STR00128##
[0247] EXAMPLE. (3,4), (5,6)-Bisacetonide-D-Gluconic Acid Methyl
Ester. In a dry 250 mL round bottom flask, under argon
S-gluconolactone (4.14 g, 23.24 mmol) was suspended in
acetone-methanol (50 mL). To this suspension dimethoxypropane
(17.15 mL, 139.44 mmol) followed by catalytic amount of
p-toulenesulfonic acid (200 mg) were added. This solution was
stirred at room temperature for 16 h. TLC (50% EtOAc in petroleum
ether) showed that all of the starting material had been consumed
and product had been formed. Acetone-methanol was removed under
reduced pressure. The residue of the reaction was dissolved in
EtOAc and washed with water. The organic layer was washed with
brine, dried over Na.sub.2SO.sub.4, and concentrated to dryness.
This material was then loaded onto a SiO.sub.2 column and
chromatographed (30% EtOAc in petroleum ether) to yield pure (3,4),
(5.6)-bisacetonide-D-gluconic acid methyl ester (3.8 g, 56%) and
regio-isomer (2,3), (5,6)-bisacetonide-D-gluconic acid methyl ester
(0.71 g, 10%). .sup.1H NMR data was in accordance with the required
products. C.sub.13HO.sub.22O.sub.7; MW 290.31; Exact Mass:
290.14.
##STR00129##
[0248] EXAMPLE. (3,4), (5,6)-Bisacetonide-2-OTf-D-Gluconic Acid
Methyl Ester. In a dry 100 mL round bottom flask, under argon
(3,4), (5,6)-bisacetonide-D-gluconic acid methyl ester (3.9 g,
13.43 mmol) was dissolved in methylene chloride (40 mL) and cooled
to -20.degree. C. to -25.degree. C. To this solution pyridine (3.26
mL, 40.29 mmol) followed by triflic anhydride (3.39 mL, 20.15 mmol)
were added. This white turbid solution was stirred at -20.degree.
C. for 1 h. TLC (25% EtOAc in petroleum ether) showed that all of
the starting material had been consumed and product had been
formed. The reaction mixture was poured into crushed-ice and
extracted with diethyl ether. The organic layer was washed with
water, brine, dried over Na.sub.2SO.sub.4, and concentrated to
yield (3,4), (5,6)-bisacetonide-2-OTf-D-gluconic acid methyl ester
(5.5 g, 97%). This material was used in the next reaction without
further purification. C.sub.14H.sub.21F.sub.3O.sub.9S MW 422.37;
Exact Mass: 422.09.
##STR00130##
[0249] EXAMPLE. (3,4), (5,6)-Bisacetonide-2-Deoxy-Azido-D-Mannonic
Acid Methyl Ester. In a dry 100 mL round bottom flask, under argon
(3,4), (5,6)-bisacetonide-2-OTf-D-gluconic acid methyl ester (5.5 g
g, 13.02 mmol) was dissolved in DMF (20 mL). To this solution
NaN.sub.3 (0.93 g, 14.32 mmol) was added. This solution was stirred
at room temperature for 1 h. TLC (8% EtOAc in petroleum ether,
triple run) showed that all of the starting material had been
consumed and product had been formed. DMF was removed under reduced
pressure. The reaction mixture was diluted with brine and extracted
with EtOAc. The organic layer was washed with water, brine, dried
over Na.sub.2SO.sub.4, and concentrated to dryness. This crude
material was then loaded onto a SiO.sub.2 column and
chromatographed (20% EtOAc in petroleum ether) to yield pure (3,4),
(5,6)-bisacetonide-2-deoxy-2-azido-D-mannonic acid methyl ester
(3.8 g, 93%). .sup.1H NMR data was in accordance with the product.
C.sub.13H.sub.21N.sub.3O.sub.6; MW 315.32; Exact Mass: 315.14.
##STR00131##
[0250] EXAMPLE. (3,4),
(5,6)-Bisacetonide-2-Deoxy-2-Amino-D-Mannonic Acid Methyl Ester. In
a Parr hydrogenation flask, (3,4),
(5,6)-bisacetonide-2-deoxy-2-azido-D-mannonic acid methyl ester
(3.5 g g, 11.10 mmol) was dissolved in methanol (170 mL). To this
solution 10% Pd on carbon (800 mg, 5 mol %) was added.
Hydrogenation was carried out using Parr-hydrogenator at 25 PSI for
1 h. TLC (10% methanol in methylene chloride) showed that all of
the starting material had been consumed and product had been
formed. The reaction mixture was filtered through a celite pad and
concentrated to dryness. This crude material was then loaded onto a
SiO.sub.2 column and chromatographed (2% methanol in methylene
chloride) to yield pure (3,4),
(5,6)-bisacetonide-2-deoxy-2-amino-D-mannonic acid methyl ester
(2.61 g, 81%). .sup.1H NMR data was in accordance with the product.
C.sub.13H.sub.23NO.sub.6S MW 289.32; Exact Mass: 289.15.
##STR00132##
[0251] EXAMPLE. (3,4),
(5,6)-Bisacetonide-2-Deoxy-2-Fmoc-Amino-D-Mannonic Acid. In a dry
100 mL, round bottom flask, (3,4),
(5,6)-bisacetonide-2-deoxy-2-amino-D-mannonic acid methyl ester
(1.24 g, 4.29 mmol) was dissolved in THF/MeOH (20 mL/5 mL). To this
solution LiOH.H.sub.2O (215.8 mg, 5.14 mmol) in water (5 mL) was
added. This light yellow solution was stirred at room temperature
for 2 h. TLC (10% methanol in methylene chloride) showed that all
of the starting material had been consumed and product had been
formed. THF/MeOH was removed under reduced pressure. The aqueous
phase was re-suspended in sat. NaHCO.sub.3 (10 mL). To this
suspension Fmoc-OSu (1.74 g, 5.14 mmol) in 1,4-dioxane (10 mL) was
added. This heterogeneous solution was stirred at room temperature
for 18 h. TLC (10% methanol in methylene chloride) showed that most
of the starting material had been consumed and product had been
formed. Dioxane was removed under reduced pressure. The aqueous
layer was extracted with diethyl ether to remove less polar
impurities. Then the aqueous layer was acidified to pH 6 using 0.2N
HCl, and re-extracted with EtOAc. The EtOAc layer was washed with
brine, dried over Na.sub.2SO.sub.4, and concentrated to yield
(3,4), (5,6)-bisacetonide-2-deoxy-2-Fmoc-amino-D-mannonic acid (1.6
g, 76%). This material was used in the next reaction without
further purification. H NMR data was in accordance with the
product. C.sub.27H.sub.31NO.sub.8; MW 497.54; Exact Mass:
497.20.
##STR00133##
[0252] EXAMPLE. EC0233 was synthesized by SPPS in three steps
according to the general peptide synthesis procedure described
herein starting from H-Cys(4-methoxytrityl)-2-chlorotrityl-Resin,
and the following SPPS reagents:
TABLE-US-00007 Reagents mmol Equivalent MW amount
H-Cys(4-methoxytrityl)- 0.56 1.0 g 2-chlorotrityl-Resin (loading
0.56 mmol/g) (3,4),(5,6)-bisacetonidc- 0.7 1.25 497.54 0.348 g
2-dcoxy-2-Fmoc-amino- D-mannonic acid Fmoc-Glu-OtBu 1.12 2 425.5
0.477 g N.sup.10TFA-Pteroic Acid 0.70 1.25 408 0.286 g (dissolve in
10 ml DMSO) DIPEA 2.24 4 129.25 0.390 mL (d = 0.742) PyBOP 1.12 2
520 0.583 g
[0253] Coupling steps. In a peptide synthesis vessel add the resin,
add the amino acid solution, DIPEA, and PyBOP. Bubble argon for 1
hr. and wash 3.times. with DMF and IPA. Use 20% piperdine in DMF
for Fmoc deprotection, 3.times. (10 min), before each amino acid
coupling. Continue to complete all 3 coupling steps. At the end
wash the resin with 2% hydrazine in DMF 3.times. (5 min) to cleave
TFA protecting group on Pteroic acid.
[0254] Cleavage step. Cleavage Reagent: 92.5% (50 ml) TFA, 2.5%
(1.34 ml) H.sub.2O, 2.5% (1.34 ml) triisopropylsilane, 2.5% (1.34
ml) ethanedithiol. Add 25 ml cleavage reagent and bubble argon for
20 min, drain, and wash 3.times. with remaining reagent. Rotavap
until 5 ml remains and precipitate in ethyl ether. Centrifuge and
dry.
[0255] HPLC Purification step. Column: Waters NovaPak C.sub.18
300.times.19 mm; Buffer A=10 mM ammonium acetate, pH 5; B=ACN;
Method: 1% B to 20% B in 40 minutes at 15 ml/min; yield .about.202
mg, 50%. C.sub.28H.sub.35N.sub.9O.sub.12S; MW 721.70; Exact Mass:
721.21.
##STR00134##
[0256] EXAMPLE. Bis-Saccharo-Folate Linker EC0244. EC0244 was
synthesized by SPPS in five steps according to the general peptide
synthesis procedure described herein starting from
H-Cys(4-methoxytrityl)-2-chlorotrityl-Resin, and the following SPPS
reagents:
TABLE-US-00008 Reagents mmol Equivalent MW amount
H-Cys(4-methoxytrityl)-2- 0.56 1.0 g chlorotrityl-Resin (loading
0.56 mmol/g) (3,4),(5,6)-bisacetonide-2- 0.7 1.25 497.54 0.348 g
deoxy-2-Fmoc-amino-D- mannonic acid Fmoc-Asp(OtBu)-OH 1.12 2 411.5
0.461 g (3,4),(5,6)-bisacetonide-2- 0.7 1.25 497.54 0.348 g
deoxy-2-Fmoc-amino-D- mannonic acid Fmoc-Glu-OtBu 1.12 2 425.5
0.477 g N.sup.10TFA-Pteroic Acid 0.70 1.25 408 0.286 g (dissolve in
10 ml DMSO) DIPEA 2.24 4 129.25 0.390 mL (d = 0.742) PyBOP 1.12 2
520 0.583 g
[0257] The Coupling steps, Cleavage step, Cleavage Reagent, and
HPLC Purification step were identical to those described above;
yield .about.284 mg, 50%. C.sub.38H.sub.51N.sub.11O.sub.20S; MW
1013.94; Exact Mass: 1013.30.
##STR00135##
[0258] EXAMPLE. EC0257 was synthesized by SPPS in six steps
according to the general peptide synthesis procedure described
herein starting from H-Cys(4-methoxytrityl)-2-chlorotrityl-Resin,
and the following SPPS reagents:
TABLE-US-00009 Reagents mmol Equivalent MW amount
H-Cys(4-methoxytrityl)-2- 0.2 0.333 g chlorotrityl-Resin (loading
0.56 mmol/g) (3,4),(5,6)-bisacetonide-2- 0.25 1.25 497.54 0.124 g
deoxy-2-Fmoc-amino-D- mannonic acid (3,4),(5,6)-bisacetonide-2-
0.25 1.25 497.54 0.124 g deoxy-2-Fmoc-amino-D- mannonic acid
Fmoc-Asp(OtBu)-OH 0.4 2 411.5 0.165 g (3,4),(5,6)-bisacetonide-2-
0.25 1.25 497.54 0.124 g deoxy-2-Fmoc-amino-D- mannonic acid
Fmoc-Glu-OtBu 0.4 2 425.5 0.170 g N.sup.10TFA-Pteroic Acid 0.25
1.25 408 0.119 g (dissolve in 10 ml DMSO) DIPEA 0.8 4 129.25 0.139
mL (d = 0.742) PyBOP 0.4 2 520 0.208 g
[0259] The Coupling steps, Cleavage step, Cleavage Reagent, and
HPLC Purification step were identical to those described above;
yield .about.170 mg, 71%. C.sub.44H.sub.62N.sub.12O.sub.25S; MW
1191.09; Exact Mass: 1190.37.
##STR00136##
[0260] EXAMPLE. EC0261 was synthesized by SPPS in seven steps
according to the general peptide synthesis procedure described
herein starting from H-Cys(4-methoxytrityl)-2-chlorotrityl-Resin.
and the following SPPS reagents:
TABLE-US-00010 Reagents mmol equivalent MW Amount
H-Cys(4-methoxytrityl)-2- 0.2 0.333 g chlorotrityl-Resin (loading
0.56 mmol/g) (3,4),(5,6)-bisacetonide-2- 0.25 1.25 497.54 0.124 g
deoxy-2-Fmoc-amino-D- mannonic acid Fmoc-Asp(OtBu)-OH 0.4 2 411.5
0.165 g (3,4),(5,6)-bisacetonide- 0.25 1.25 497.54 0.124 g
2-deoxy-2-Fmoc-amino- D-mannonic acid Fmoc-Asp(OtBu)-OH 0.4 2 411.5
0.165 g (3,4),(5,6)-bisacetonide-2- 0.25 1.25 497.54 0.124 g
deoxy-2-Fmoc-amino-D- mannonic acid Fmoc-Glu-OtBu 0.4 2 425.5 0.170
g N.sup.10TFA-Pteroic Acid 0.25 1.25 408 0.119 g (dissolve in 10 ml
DMSO) DIPEA 0.8 4 129.25 0.139 mL (d = 0.742) PyBOP 0.4 2 520 0.208
g
[0261] The Coupling steps, Cleavage step, Cleavage Reagent, and
HPLC Purification step were identical to those described above;
yield .about.170 mg, 65%. C.sub.48H.sub.67N.sub.13O.sub.28S; MW
1306.18; Exact Mass: 1305.39
##STR00137##
[0262] EXAMPLE. Tetra-Saccharo-Tris-Asp-Folate Linker EC0268.
EC0268 was synthesized by SPPS in nine steps according to the
general peptide synthesis procedure described herein starting from
H-Cys(4-methoxytrityl)-2-chlorotrityl-Resin, and the following SPPS
reagents:
TABLE-US-00011 Reagents mmol equivalent MW Amount
H-Cys(4-methoxytrityl)-2- 0.1 0.167 g chlorotrityl-Resin (loading
0.56 mmol/g) (3,4),(5,6)-bisacetonide-2- 0.125 1.25 497.54 0.062 g
deoxy-2-Fmoc-amino-D- mannonic acid Fmoc-Asp(OtBu)-OH 0.2 2 411.5
0.082 g (3,4),(5,6)-bisacetonide-2- 0.125 1.25 497.54 0.062 g
deoxy-2-Fmoc-amino-D- mannonic acid Fmoc-Asp(OtBu)-OH 0.2 2 411.5
0.082 g (3,4),(5,6)-bisacetonide-2- 0.125 1.25 497.54 0.062 g
deoxy-2-Fmoc-amino-D- mannonic acid Fmoc-Asp(OtBu)-OH 0.2 2 411.5
0.082 g (3,4),(5,6)-bisacetonide-2- 0.125 1.25 497.54 0.062 g
deoxy-2-Fmoc-amino-D- mannonic acid Fmoc-Glu-OtBu 0.2 2 425.5 0.085
g N.sup.10TFA-Pteroic Acid 0.125 1.25 408 0.059 g (dissolve in 10
ml DMSO) DIPEA 0.4 4 129.25 0.070 mL (d = 0.742) PyBOP 0.2 2 520
0.104 g
[0263] The Coupling steps, Cleavage step, Cleavage Reagent, and
HPLC Purification step were identical to those described above;
yield .about.100 mg, 63%. C.sub.94H.sub.125N.sub.19O.sub.37S.sub.2;
MW 2177.24; Exact Mass: 2175.79.
[0264] The following illustrative examples may be prepared
according to the procedure for EC0268:
##STR00138##
[0265] EXAMPLE. Tetra-Saccharo-Asp-Folate Linker EC0463. EC0463 was
synthesized by SPPS in seven steps according to the general peptide
synthesis procedure described herein starting from
H-Cys(4-methoxytrityl)-2-chlorotrityl-Resin, and the following SPPS
reagents:
TABLE-US-00012 Reagents mmol equivalent MW Amount
H-Cys(4-methoxytrityl)-2- 0.1 0.167 g chlorotrityl-Resin (loading
0.56 mmol/g) (3,4),(5,6)-bisacetonide-2- 0.125 1.25 497.54 0.062 g
deoxy-2-Fmoc-amino-D- mannonic acid (3,4),(5,6)-bisacetonide-2-
0.125 1.25 497.54 0.062 g deoxy-2-Fmoc-amino-D- mannonic acid
(3,4),(5,6)-bisacetonide-2- 0.125 1.25 497.54 0.062 g
deoxy-2-Fmoc-amino-D- mannonic acid Fmoc-Asp(OtBu)-OH 0.2 2 411.5
0.082 g (3,4),(5,6)-bisacetonide-2- 0.125 1.25 497.54 0.062 g
deoxy-2-Fmoc-amino-D- mannonic acid Fmoc-Glu-OtBu 0.2 2 425.5 0.085
g N.sup.10TFA-Pteroic Acid 0.125 1.25 408 0.059 g (dissolve in 10
ml DMSO) DIPEA 0.4 4 129.25 0.070 mL (d = 0.742) PyBOP 0.2 2 520
0.104 g
[0266] The Coupling steps, Cleavage step, Cleavage Reagent, and
HPLC Purification step were identical to those described above;
yield .about.63 mg, 46%. C.sub.50H.sub.73N.sub.13O.sub.30S; MW
1368.25; Exact Mass: 1367.43.
##STR00139##
[0267] EXAMPLE. Tetra-Saccharo-Bis-.alpha.-Glu-Arg-Folate Linker
EC0480. EC0480 was synthesized by SPPS in nine steps according to
the general peptide synthesis procedure described herein starting
from H-Cys(4-methoxytrityl)-2-chlorotrityl-Resin, and the following
SPPS reagents:
TABLE-US-00013 Reagents mmol equivalent MW Amount
H-Cys(4-methoxytrityl)-2- 0.2 0.333 g chlorotrityl-Resin (loading
0.56 mmol/g) (3,4),(5,6)-bisacetonide-2- 0.250 1.25 497.54 0.124 g
deoxy-2-Fmoc-amino-D- mannonic acid Fmoc-Glu(OtBu)-OH 0.4 2 425.5
0.170 g (3,4),(5,6)-bisacetonide-2- 0.250 1.25 497.54 0.124 g
deoxy-2-Fmoc-amino-D- mannonic acid Fmoc-Arg(Pbf)-OH 0.4 2 648.78
0.260 g (3,4),(5,6)-bisacetonide-2- 0.250 1.25 497.54 0.124 g
deoxy-2-Fmoc-amino-D- mannonic acid Fmoc-Glu(OtBu)-OH 0.4 2 425.5
0.170 g (3,4),(5,6)-bisacetonide-2- 0.250 1.25 497.54 0.124 g
deoxy-2-Fmoc-amino-D- mannonic acid Fmoc-Glu-OtBu 0.4 2 425.5 0.170
g N.sup.10TFA-Pteroic Acid 0.250 1.25 408 0.119 g (dissolve in 10
ml DMSO) DIPEA 0.8 4 129.25 0.139 mL (d = 0.742) PyBOP 0.4 2 520
0.208 g
[0268] The Coupling steps, Cleavage step, Cleavage Reagent, and
HPLC Purification step were identical to those described above;
yield .about.100 mg, 33%. C.sub.62H.sub.94N.sub.18O.sub.20S; MW
1667.58; Exact Mass: 1666.59.
[0269] EXAMPLE. Tetra-Saccharo-Bis-Asp-Folate Linker EC0452:
##STR00140##
[0270] EXAMPLE. Tetra-Saccharo-Bis-Asp-Folate Linker EC0452. EC0452
was synthesized by SPPS in nine steps according to the general
peptide synthesis procedure described herein starting from
H-Cys(4-methoxytrityl)-2-chlorotrityl-Resin, and the following
SPPS-reagents:
TABLE-US-00014 Reagents mmol equivalent MW Amount
H-Cys(4-methoxytrityl)-2- 0.15 0.250 g chlorotrityl-Resin (loading
0.6 mmol/g) (3,4),(5,6)-bisacetonide-2- 0.188 1.25 497.54 0.094 g
deoxy-2-Fmoc-amino-D- mannonic acid Fmoc-Asp(OtBu)-OH 0.3 2 411.5
0.123 g (3,4),(5,6)-bisacetonide-2- 0.188 1.25 497.54 0.094 g
deoxy-2-Fmoc-amino-D- mannonic acid Fmoc-4-(2-aminoethyl)-1- 0.3 2
482.42 0.145 g carboxymethyl-piperazine dihydrochloride
(3,4),(5,6)-bisacetonide-2- 0.188 1.25 497.54 0.094 g
deoxy-2-Fmoc-amino-D- mannonic acid Fmoc-Asp(OtBu)-OH 0.3 2 411.5
0.123 g (3,4),(5,6)-bisacetonide-2- 0.188 1.25 497.54 0.094 g
deoxy-2-Fmoc-amino-D- mannonic acid Fmoc-Glu-OtBu 0.3 2 425.5 0.128
g N.sup.10TFA-Pteroic Acid 0.188 1.25 408 0.077 g (dissolve in 10
ml DMSO) DIPEA 0.6 4 129.25 0.105 mL (d = 0.742) PyBOP 0.3 2 520
0.156 g
[0271] The Coupling steps, Cleavage step, and Cleavage Reagent were
identical to those described above. HPLC Purification step. Column:
Waters NovaPak C.sub.18 300.times.19 mm; Buffer A=10 mM ammonium
acetate, pH 5; B=ACN; Method: 1% B to 20% B in 40 minutes at 25
ml/min; yield .about.98 mg, 40%. C.sub.62H.sub.93N.sub.17O.sub.34S;
MW 1652.56; Exact Mass: 1651.58.
##STR00141##
[0272] EXAMPLE. Tetra-Saccharo-bis-Asp-Folate Linker EC0457. EC0457
was synthesized by SPPS in eight steps according to the general
peptide synthesis procedure described herein starting from
H-Cys(4-methoxytrityl)-2-chlorotrityl-Resin, and the following SPPS
reagents:
TABLE-US-00015 Reagents mmol equivalent MW Amount
H-Cys(4-methoxytrityl)-2- 0.20 0.333 g chlorotrityl-Resin (loading
0.6 mmol/g) (3,4),(5,6)-bisacetonide-2- 0.25 1.25 497.54 0.124 g
deoxy-2-Fmoc-amino-D- mannonic acid Fmoc-Asp(OtBu)-OH 0.30 1.5
411.5 0.123 g (3,4),(5,6)-bisacetonide-2- 0.25 1.25 497.54 0.124 g
deoxy-2-Fmoc-amino-D- mannonic acid (3,4),(5,6)-bisacetonide-2-
0.25 1.25 497.54 0.124 g deoxy-2-Fmoc-amino-D- mannonic acid
Fmoc-Asp(OtBu)-OH 0.30 1.5 411.5 0.123 g
(3,4),(5,6)-bisacetonide-2- 0.25 1.25 497.54 0.124 g
deoxy-2-Fmoc-amino-D- mannonic acid Fmoc-Glu-OtBu 0.30 1.5 425.5
0.128 g N.sup.10TFA-Pteroic Acid 0.25 1.25 408 0.102 g (dissolve in
10 ml DMSO) DIPEA 2 eq. of 129.25 amino acid (d = 0.742) 87 .mu.L
or 105 .mu.L PyBOP 2 eq. of 520 260 mg or amino acid 312 mg
[0273] The Coupling steps, Cleavage step, and Cleavage Reagent were
identical to those described above. HPLC Purification step. Column:
Waters NovaPak C.sub.18 300.times.19 mm; Buffer A=10 mM ammonium
acetate, pH 5; B=ACN; Method: 0% B to 20% B in 40 minutes at 25
ml/min; yield 210 mg, 71%. C.sub.54H.sub.78N.sub.14O.sub.11S; MW
1483.34; Exact Mass: 1482.46.
##STR00142##
[0274] EXAMPLE. Tetra-Saccharo-tris-Glu-Folate Linker EC0477.
EC0477 was synthesized by SPPS in nine steps according to the
general peptide synthesis procedure described herein starting from
H-Cys(4-methoxytrityl)-2-chlorotrityl-Resin, and the following SPPS
reagents:
TABLE-US-00016 Reagents mmol equivalent MW Amount
H-Cys(4-methoxytrityl)-2- 0.20 0.333 g chlorotrityl-Resin (loading
0.6 mmol/g) (3,4),(5,6)-bisacetonide-2- 0.25 1.25 497.54 0.124 g
deoxy-2-Fmoc-amino-D- mannonic acid Fmoc-Glu(OtBu)-OH 0.30 1.5
425.5 0.128 g (3,4),(5,6)-bisacetonide-2- 0. 25 1.25 497.54 0.124 g
deoxy-2-Fmoc-amino-D- mannonic acid Fmoc-Glu(OtBu)-OH 0.30 1.5
425.5 0.128 g (3,4),(5,6)-bisacetonide-2- 0.25 1.25 497.54 0.124 g
deoxy-2-Fmoc-amino-D- mannonic acid Fmoc-Glu(OtBu)-OH 0.30 1.5
425.5 0.128 g (3,4),(5,6)-bisacetonide-2- 0.25 1.25 497.54 0.124 g
deoxy-2-Fmoc-amino-D- mannonic acid Fmoc-Glu-OtBu 0.30 1.5 425.5
0.128 g N.sup.10TFA-Pteroic Acid 0.25 1.25 408 0.102 g (dissolve in
10 ml DMSO) DIPEA 2 eq. of 129.25 87 .mu.L or amino acid (d =
0.742) 105 .mu.L PyBOP 1 eq. of 520 130 mg or amino acid 156 mg
[0275] The Coupling steps, Cleavage step, and Cleavage Reagent were
identical to those described above. HPLC Purification step. Column:
Waters NovaPak C.sub.18 300.times.19 mm; Buffer A=10 mM ammonium
acetate. pH 5; B=ACN; Method: 0% B to 20% B in 40 minutes at 25
ml/min; yield 220 mg, 67%. C.sub.61H.sub.89N.sub.15O.sub.36S; MW
1640.50; Exact Mass: 1639.53.
##STR00143##
[0276] EXAMPLE. EC0453 was synthesized by SPPS according to the
general peptide synthesis procedure described herein starting from
H-Cys(4-methoxytrityl)-2-chlorotrityl-Resin, and the following SPPS
reagents:
TABLE-US-00017 Reagents mmol equivalent MW Amount
H-Cys(4-methoxytrityl)-2- 0.162 0.290 g chlorotrityl-Resin (loading
0.56 mmol/g) (3,4),(5,6)-bisacetonide-2- 0.203 1.25 497.54 0.101 g
deoxy-2-Fmoc-amino-D- mannonic acid Fmoc-Asp(OtBu)-OH 0.324 2 411.5
0.133 g (3,4),(5,6)-bisacetonide-2- 0.203 1.25 497.54 0.101 g
deoxy-2-Fmoc-amino-D- mannonic acid Fmoc-Asp(OtBu)-OH 0.324 2 411.5
0.133 g (3,4),(5,6)-bisacetonide-2- 0.203 1.25 497.54 0.101 g
deoxy-2-Fmoc-amino-D- mannonic acid Fmoc-Asp(OtBu)-OH 0.324 2 411.5
0.133 g (3,4),(5,6)-bisacetonide-2- 0.203 1.25 497.54 0.101 g
deoxy-2-Fmoc-amino-D- mannonic acid Fmoc-Glu-OtBu 0.324 2 425.5
0.138 g N.sup.10TFA-Pteroic Acid 0.203 1.25 408 0.083 g (dissolve
in 10 ml DMSO) DIPEA 2 eq of 71 .mu.L or AA 85 .mu.L PyBOP 1 eq of
211 mg or AA 253 mg
[0277] Coupling steps. In a peptide synthesis vessel add the resin,
add the amino acid solution, DIPEA, and PyBOP. Bubble argon for 1
hr. and wash 3.times. with DMF and IPA. Use 20% piperidine in DMF
for Fmoc deprotection, 3.times. (10 min), before each amino acid
coupling. Continue to complete all 9 coupling steps. At the end
treat the resin with 2% hydrazine in DMF 3.times. (5 min) to cleave
TFA protecting group on Pteroic acid, wash the resin with DMF
(3.times.), IPA (3.times.), MeOH (3.times.), and bubble the resin
with argon for 30 min.
[0278] Cleavage step. Cleavage Reagent: 92.5% TFA, 2.5% H.sub.2O,
2.5% triisopropylsilane, 2.5% ethanedithiol. Treat the resin with
cleavage reagent 3 times (15 min, 5 min, 5 min) with argon
bubbling, drain, collect, and combine the solution. Rotavap until 5
ml remains and precipitate in diethyl ether (35 mL). Centrifuge,
wash with diethyl ether, and dry. The crude solid was purified by
HPLC.
[0279] HPLC Purification step. Column: Waters Xterra Prep MS Cis 10
.mu.m 19.times.250 mm; Solvent A: 10 mM ammonium acetate, pH 5;
Solvent B: ACN; Method: 5 min 0% B to 40 min 20% B 25 mL/min;
Fractions containing the product was collected and freeze-dried to
give .about.60 mg EC0453 (23% yield). .sup.1H NMR and LC/MS were
consistent with the product. C.sub.58H.sub.83N.sub.15O.sub.36S; MW
1598.43; Exact Mass: 1597.48. C, 43.58; H, 5.23; N, 13.14; O,
36.03; S, 2.01.
##STR00144##
[0280] EXAMPLE. (3,4),
(5,6)-Bisacetonide-2-deoxy-2-Fmoc-amino-D-Mannonic
acid-diazo-ketone. In a dry 100 mL round bottom flask, (3,4),
(5,6)-bisacetonide-2-deoxy-2-Fmoc-amino-D-mannonic acid (1.0 g,
2.01 mmol) was dissolved in THF (10 mL, not fully dissolved) under
Argon atmosphere. The reaction mixture was cooled to -25.degree. C.
To this solution NMM (0.23 mL, 2.11 mmol) and ethylchloroformate
(228.98 mg, 2.11 mmol) were added. This solution was stirred at
-20.degree. C. for 30 min. The resulting white suspension was
allowed to warm to 0.degree. C., and a solution of diazomethane in
ether was added until yellow color persisted. Stirring was
continued as the mixture was allowed to warm to room temperature.
Stirred for 2 h, excess diazomethane was destroyed by the addition
of few drops of acetic acid with vigorous stirring. The mixture was
diluted with ether, washed with sat, aq. NaHCO.sub.3 solution, sat.
aq. NH.sub.4Cl, brine, dried over Na.sub.2SO.sub.4, and
concentrated to dryness. This crude material was then loaded onto a
SiO.sub.2 column and chromatographed (30% EtOAc in petroleum ether)
to yield pure (3,4),
(5,6)-bisacetonide-2-deoxy-2-Fmoc-amino-D-mannonic
acid-diazo-ketone (0.6 g, 57%). .sup.1H NMR data was in accordance
with the product. C.sub.2H.sub.31N.sub.3O.sub.7; MW 521.56; Exact
Mass: 521.22.
##STR00145##
[0281] EXAMPLE. (3R, 4R, 5S,
6R)-(4,5),(6,7)-Bisacetonide-3-Fmoc-Amino-Heptanoic acid. In a dry
25 mL round bottom flask, (3,4),
(5,6)-bisacetonide-2-deoxy-2-Fmoc-amino-D-mannonic
acid-diazo-ketone (0.15 g, 0.29 mmol) was dissolved in THF (1.6 mL)
under Argon atmosphere. To this solution silver trifluoroacetate
(6.6 mg, 0.03 mmol) in water (0.4 mL) was added in the dark. The
resulting mixture was stirred at room temperature for 16 h. TLC
(10% MeOH in methylene chloride) showed that all of the starting
material had been consumed and product had been formed. Solvent
(THF) was removed under reduced pressure, the residue was diluted
with water (pH was 3.5-4.0) and extracted with EtOAc. The organic
layer was washed with brine, dried over Na.sub.2SO.sub.4, and
concentrated to dryness. This crude material was then loaded unto a
SiO.sub.2 column and chromatographed (gradient elution from 1% MeOH
in methylene chloride to 5% MeOH in methylene chloride) to yield
pure (3R, 4R, 5S,
6R)-(4,5),(6,7)-bisacetonide-3-Fmoc-amino-heptanoic acid (0.10 g,
68%). .sup.1H NMR data was in accordance with the product.
C.sub.28H.sub.33NO.sub.8; MW 511.56; Exact Mass: 511.22.
##STR00146##
[0282] EXAMPLE. Tetra-Homosaccharo-Tris-.alpha.Glu-Folate Spacer
EC0478. EC0478 was synthesized by SPPS in nine steps according to
the general peptide synthesis procedure described herein starting
from H-Cys(4-methoxytrityl)-2-chlorotrityl-Resin, and the following
SPPS reagents:
TABLE-US-00018 Reagents mmol equivalent MW Amount
H-Cys(4-methoxytrityl)-2- 0.1 0.167 g chlorotrityl-Resin (loading
0.56 mmol/g) Homo sugar 0.12 1.2 511.56 0.061 g Fmoc-Glu(OtBu)-OH
0.2 2 425.5 0.085 g Homo sugar 0.12 1.2 511.56 0.061 g
Fmoc-Glu(OtBu)-OH 0.2 2 425.5 0.085 g Homo sugar 0.12 1.2 511.56
0.061 g Fmoc-Glu(OtBu)-OH 0.2 2 425.5 0.085 g Homo sugar 0.12 1.2
511.56 0.061 g Fmoc-Glu-OtBu 0.2 2 425.5 0.085 g
N.sup.10TFA-Pteroic Acid.cndot.TFA 0.12 1.2 408 0.049 g (dissolve
in 10 ml DMSO) DIPEA 0.4 4 129.25 0.070 mL (d = 0.742) PyBOP 0.2 2
520 0.104 g
[0283] The Coupling steps, Cleavage step, and Cleavage Reagent were
identical to those described above. HPLC Purification step: Column:
Waters NovaPak C.sub.18 300.times.19 mm; Buffer A=10 mM ammonium
acetate, pH 5; B=ACN; Method: 100% A for 5 min then 0% B to 20% B
in 20 minutes at 26 ml/min; yield .about.88 mg, 52%.
C.sub.65H.sub.97N.sub.15O.sub.36S; MW 1696.61; Exact Mass:
1695.59.
##STR00147##
[0284] EXAMPLE. (3,4), (5,6)-Bisacetonide-D-Gluconic Amide. 20 g of
the methyl ester was dissolved in 100 mL methanol, cooled the
high-pressure reaction vessel with dry ice/acetone, charged with
100 mL liquid ammonia, warmed up to room temperature and heated to
160.degree. C./850 PSI for 2 hours. The reaction vessel was cooled
to room temperature and released the pressure. Evaporation of the
solvent gave brownish syrup, and minimum amount of isopropyl
alcohol was added to make the homogeneous solution with reflux. The
solution was cooled to -20.degree. C. and the resulting solid was
filtered to give 8.3 g of solid. The mother liquid was evaporated,
and to the resulting residue, ether was added and refluxed until
homogeneous solution was achieved. The solution was then cooled to
-20.degree. C. and the resulting solid was filtered to give 4.0 g
product. The solid was combined and recrystallized in isopropyl
alcohol to give 11.2 g (59%) of the white amide product
C.sub.12H.sub.21NO.sub.4; MW 275.30; Exact Mass: 275.14.
##STR00148##
[0285] EXAMPLE. (3,4),
(5,6)-Bisacetonide-1-Deoxy-1-Amino-D-Glucitol. In a dry 100 mL
round bottom flask, under argon, LiAlH.sub.4 (450 mg, 11.86 momol))
was dissolved in THF (10 mL) and cooled to 0.degree. C. To this
suspension (3,4), (5,6)-bisacetonide-D-gluconic amide (1.09 g, 3.96
mmol) in THF (30 mL) was added very slowly over 15 min. This
mixture was refluxed for 5 h. TLC (10% MeOH in methylene chloride)
showed that all of the starting material had been consumed and
product had been formed. The reaction mixture was cooled to room
temperature, and then cooled to ice-bath temperature, diluted with
diethyl ether (40 mL), slowly added 0.5 mL of water, 0.5 mL of 15%
aq. NaOH, and then added 1.5 mL of water. The reaction mixture was
warmed to room temperature and stirred for 30 min. MgSO.sub.4 was
added and stirred for additional 15 min and filtered. The organic
layer was concentrated to dryness to yield (3,4),
(5,6)-bisacetonide-1-deoxy-1-amino-D-glucitol. H NMR data was in
accordance with the product. C.sub.12H.sub.23NO.sub.5; MW 261.31;
Exact Mass: 261.16.
##STR00149##
[0286] EXAMPLE. EC0475. O-Allyl protected Fmoc-Glu (2.17 g, 1 eq),
PyBOP (2.88 g, 1 eq), and DIPEA (1.83 mL, 2 eq) were added to a
solution of (3,4),(5,6)-bisacetonide-1-deoxy-1-amino-D-glucitol
(1.4 g, 5.3 mmol) in dry DMF (6 mL) and the mixture was stirred at
RT under Ar for 2 h. The solution was diluted with EtOAc (50 mL),
washed with brine (10 mL.times.3), organic layer separated, dried
(MgSO.sub.4), filtered and concentrated to give a residue, which
was purified by a flash column (silica gel, 60% EtOAc/petro-ether)
to afford 1.72 g (50%) allyl-protected EC0475 as a solid.
Pd(Ph.sub.3).sub.4 (300 mg, 0.1 eq) was added to a solution of
allyl-protected EC0475 (1.72 g, 2.81 mmol) in NMM/AcOH/CHCl.sub.3
(2 mL/4 mL/74 mL). The resulting yellow solution was stirred at RT
under Ar for 1 h, to which was added a second portion of
Pd(Ph.sub.3).sub.4 (300 mg, 0.1 eq). After stirring for an
additional 1 h, the mixture was washed with 1 N HCl (50 mL.times.3)
and brine (50 mL), organic layer separated, dried (MgSO4),
filtered, and concentrated to give a yellow foamy solid, which was
subject to chromatography (silica gel, 1% MeOH/CHCl.sub.3 followed
by 3.5% MeOH/CHCl.sub.3) to give 1.3 g (81%) EC0475 as a solid
material. MW 612.67; Exact Mass: 612.27.
##STR00150##
[0287] EXAMPLE. Tetra-Saccharoglutamate-Bis-.alpha.Glu-Folate
Spacer EC0491. EC0491 was synthesized by SPPS in eight steps
according to the general peptide synthesis procedure described
herein starting from H-Cys(4-methoxytrityl)-2-chlorotrityl-Resin,
and the following SPPS reagents:
TABLE-US-00019 Reagents Mmol equivalent MW Amount
H-Cys(4-methoxytrityl)-2- 0.1 0.167 g chlorotrityl-Resin (loading
0.56 mmol/g) EC0475 0.13 1.3 612.67 0.080 g Fmoc-Glu(OtBu)-OH 0.2 2
425.5 0.085 g EC0475 0.13 1.3 612.67 0.080 g EC0475 0.13 1.3 612.67
0.080 g Fmoc-Glu(OtBu)-OH 0.2 2 425.5 0.085 g EC0475 0.13 1.3
612.67 0.080 g Fmoc-Glu-OtBu 0.2 2 425.5 0.085 g
N.sup.10TFA-Pteroic Acid.cndot.TFA 0.2 2 408 0.105 g (dissolve in
10 ml DMSO) DIPEA 0.4 4 129.25 0.070 mL (d = 0.742) PyBOP 0.2 2 520
0.104 g
[0288] The Coupling steps, Cleavage step, and Cleavage Reagent were
identical to those described above. HPLC Purification step: Column:
Waters NovaPak C.sub.1 300.times.19 mm; Buffer A=10 mM ammonium
acetate, pH 5; B=ACN; Method: 100% A for 5 min then 0% B to 20% B
in 20 minutes at 26 ml/min; yield 100 mg, 51%.
C.sub.76H.sub.118N.sub.18O.sub.41S; MW 1971.91; Exact Mass:
1970.74.
##STR00151##
[0289] EXAMPLE. EC04/9 was synthesized by SPPS according to the
genera peptide synthesis procedure described herein starting from
H-Cys(4-methoxytrityl)-2-chlorotrityl-Resin, and the following SPPS
reagents:
TABLE-US-00020 Reagents mmol equivalent MW Amount
H-Cys(4-methoxytrityl)-2- 0.094 0.16 g chlorotrityl-Resin (loading
0.6 mmol/g) EC0475 0.13 1.4 612.67 0.082 g Fmoc-Glu(OtBu)-OH 0.19
2.0 425.47 0.080 g EC0475 0.13 1.4 612.67 0.082 g Fmoc-Arg(Pbf)-OH
0.19 2.0 648.77 0.12 g EC0475 0.13 1.4 612.67 0.082 g
Fmoc-Glu(OtBu)-OH 0.19 2.0 425.47 0.080 g EC0475 0.13 1.4 612.67
0.082 g Fmoc-Glu-OtBu 0.19 2.0 425.47 0.080 g N.sup.10TFA-Pteroic
Acid 0.16 1.7 408.29 0.066 g (dissolve in 10 ml DMSO) DIPEA 2.0 eq
of 41 .mu.L or AA 49 .mu.L PyBOP 1.0 eq of 122 mg or AA 147 mg
[0290] Coupling steps. In a peptide synthesis vessel add the resin,
add the amino acid solution, DIPEA, and PyBOP. Bubble argon for 1
hr. and wash 3.times. with DMF and IPA. Use 20% piperidine in DMF
for Fmoc deprotection, 3.times. (10 min), before each amino acid
coupling. Continue to complete all 9 coupling steps. At the end
treat the resin with 2% hydrazine in DMF 3.times. (5 min) to cleave
TFA protecting group on Pteroic acid, wash the resin with DMF
(3.times.), IPA (3.times.), MeOH (3.times.), and bubble the resin
with argon for 30 min.
[0291] Cleavage step. Reagent: 92.5% TFA, 2.5% H.sub.2O, 2.5%
triisopropylsilane, 2.5% ethanedithiol. Treat the resin with
cleavage reagent for 15 min with argon bubbling, drain, wash the
resin once with cleavage reagent, and combine the solution. Rotavap
until 5 ml remains and precipitate in diethyl ether (35 mL).
Centrifuge, wash with diethyl ether, and dry. The crude solid was
purified by HPLC.
[0292] HPLC Purification step. Column: Waters Atlantis Prep T3 10
.mu.m OBD 19.times.250 mm; Solvent A: 10 mM ammonium acetate, pH 5;
Solvent B: ACN; Method: 5 min 0% B to 20 min 20% B 26 mL/min.
Fractions containing the product was collected and freeze-dried to
give .about.70 mg EC0479 (35% yield). .sup.1H NMR and LC/MS were
consistent with the product. MW 2128.10; Exact Mass: 2126.84.
##STR00152##
[0293] EC0488. This compound was prepared by SPPS according to the
general peptide synthesis procedure described herein starting from
H-Cys(4-methoxytrityl)-2-chlorotrityl-Resin, and the following SPPS
reagents:
TABLE-US-00021 Reagents mmol equivalent MW amount
H-Cys(4-methoxytrityl)-2- 0.10 0.17 g chlorotrityl-Resin (loading
0.6 mmol/g) EC0475 0.13 1.3 612.67 0.082 g Fmoc-Glu(OtBu)-OH 0.19
1.9 425.47 0.080 g EC0475 0.13 1.3 612.67 0.082 g Fmoc-Glu(OtBu)-OH
0.19 1.9 425.47 0.080 g EC0475 0.13 1.3 612.67 0.082 g
Fmoc-Glu-OtBu 0.19 1.9 425.47 0.080 g N.sup.10TFA-Pteroic Acid 0.16
1.6 408.29 0.066 g (dissolve in 10 ml DMSO) DIPEA 2.0 eq of AA
PyBOP 1.0 eq of AA
[0294] Coupling steps. In a peptide synthesis vessel add the resin,
add the amino acid solution, DIPEA, and PyBOP. Bubble argon for 1
hr. and wash 3.times. with DMF and IPA. Use 20% piperidine in DMF
for Fmoc deprotection, 3.times. (10 min), before each amino acid
coupling. Continue to complete all 9 coupling steps. At the end
treat the resin with 2% hydrazine in DMF 3.times. (5 min) to cleave
TFA protecting group on Pteroic acid, wash the resin with DMF
(3.times.), IPA (3.times.), MeOH (3.times.), and bubble the resin
with argon for 30 min.
[0295] Cleavage step. Reagent: 92.5% TFA, 2.5% H.sub.2O, 2.5%
triisopropylsilane, 2.5% ethanedithiol. Treat the resin with
cleavage reagent 3.times. (10 min, 5 min, 5 min) with argon
bubbling, drain, wash the resin once with cleavage reagent, and
combine the solution. Rotavap until 5 ml remains and precipitate in
diethyl ether (35 mL). Centrifuge, wash with diethyl ether, and
dry. About half of the crude solid (.about.100 mg) was purified by
HPLC.
[0296] HPLC Purification step. Column: Waters Xterra Prep MS C18 10
.mu.m 19.times.250 mm; Solvent A: 10 mM ammonium acetate, pH 5;
Solvent B: ACN; Method: 5 min 0% B to 25 min 20% B 26 mL/min.
Fractions containing the product was collected and freeze-dried to
give 43 mg EC0488 (51% yield). .sup.1H NMR and LC/MS (exact mass
1678.62) were consistent with the product. MW 1679.63; Exact Mass:
1678.62.
[0297] The following Examples of binding ligand-linker
intermediates, EC0233, EC0244, EC0257, and EC0261, were prepared as
described herein.
##STR00153##
[0298] The following Examples of illustrative intermediates were
prepared as described herein.
##STR00154##
[0299] Huisgen azide for forming 1,2,3-triazole; (a) NaN.sub.3; (b)
Ag.sub.2CO.sub.3, DCM, molecular sieves; (c)
##STR00155## ##STR00156##
##STR00157##
[0300] EXAMPLE. Synthesis of Coupling Reagent EC0311. DIPEA (0.60
mL) was added to a suspension of
HOBt-OCO.sub.2--(CH.sub.2).sub.2--SS-2-pyridine HCl (685 mg. 91%)
in anhydrous DCM (5.0 mL) at 0.degree. C., stirred under argon for
2 minutes, and to which was added anhydrous hydrazine (0.10 mL).
The reaction mixture was stirred under argon at 0.degree. C. for 10
minutes and room temperature for an additional 30 minutes,
filtered, and the filtrate was purified by flash chromatography
(silica gel, 2% MeOH in DCM) to afford EC0311 as a clear thick oil
(371 mg), solidified upon standing.
##STR00158##
[0301] EXAMPLE. Vinblastine Pyridinyl Disulfide.
2-[(Benzotriazole-1-yl-(oxycarbonyloxy)-ethyldisulfanyl]-pyridine
HC (601 mg) and 378 .mu.L of DIPEA were sequentially added to a
solution of desacetyl vinblastine hydrazide (668 mg) in 5 ml of DCM
at 0.degree. C. The reaction was allowed to warm to room
temperature and stirred for 3 hours. TLC (15% MeOH in DCM) showed
complete conversion. The mixture was purified by silica gel
chromatography (1:9 MeOH/DCM). The combined fractions were
evaporated, redissolved in DCM and washed with 10%
Na.sub.2CO.sub.3, brine, dried (MgSO.sub.4), and evaporated to 550
mg (80%); HPLC-RT 12.651 min., 91% pure, .sup.1H HMR spectrum
consistent with the assigned structure, and MS (ESI+): 984.3,
983.3, 982.4, 492.4, 491.9, 141.8. Additional details of this
procedure are described in U.S. patent application publication No.
US 2005/0002942 A1.
##STR00159##
[0302] EXAMPLE. Preparation of Tubulysin Hydrazides. Illustrated by
preparing EC0347. N,N-Diisopropylethylamine (DIPEA, 6.1 .mu.L) and
isobutyl chloroformate (3.0 .mu.L) were added with via syringe in
tandem into a solution of tubulysin B (0.15 mg) in anhydrous EtOAc
(2.0 mL) at -15.degree. C. After stirring for 45 minutes at
-15.degree. C. under argon, the reaction mixture was cooled down to
-20.degree. C. and to which was added anhydrous hydrazine (5.0
.mu.L). The reaction mixture was stirred under argon at -20.degree.
C. for 3 hours, quenched with 10 mM sodium phosphate buffer (pH
7.0, 1.0 mL), and injected into a preparative HPLC for
purification. Column: Waters XTerra Prep MS C.sub.18 10 .mu.m,
19.times.250 mm; Mobile phase A: 1.0 mM sodium phosphate buffer, pH
7.0; Mobile phase B: acetonitrile; Method: 10% B to 80% B over 20
minutes, flow rate=25 mL/min. Fractions from 15.14-15.54 minutes
were collected and lyophilized to produce EC0347 as a white solid
(2.7 mg). The foregoing method is equally applicable for preparing
other tubulysin hydrazides by the appropriate selection of the
tubulysin starting compound.
##STR00160##
[0303] EXAMPLE. Preparation of Tubulysin Disulfides (stepwise
process). Illustrated for EC0312. DIPEA (36 .mu.L) and isobutyl
chloroformate (13 .mu.L) were added with the help of a syringe in
tandem into a solution of tubulysin B (82 mg) in anhydrous EtOAc
(2.0 mL) at -15.degree. C. After stirring for 45 minutes at
-15.degree. C. under argon, to the reaction mixture was added a
solution of EC0311 in anhydrous EtOAc (1.0 mL). The resulting
solution was stirred under argon at -15.degree. C. for 15 minutes
and room temperature for an additional 45 minutes, concentrated,
and the residue was purified by flash chromatography (silica gel, 2
to 8% MeOH in DCM) to give EC0312 as a white solid (98 mg). The
foregoing method is equally applicable for preparing other
tubulysin derivatives by the appropriate selection of the tubulysin
starting
##STR00161##
[0304] EXAMPLE. General Synthesis of Disulfide Containing Tubulysin
Conjugates. Illustrated with EC0312. A binding ligand-linker
intermediate containing a thiol group is taken in deionized water
(ca. 20 mg/mL, bubbled with argon for 10 minutes prior to use) and
the pH of the suspension was adjusted by saturated NaHCO.sub.3
(bubbled with argon for 10 minutes prior to use) to about 6.9 (the
suspension may become a solution when the pH increased). Additional
deionized water is added (ca. 20-25%) to the solution as needed,
and to the aqueous solution is added immediately a solution of
EC0312 in THF (ca. 20 mg/mL). The reaction mixture becomes
homogenous quickly. After stirring under argon, e.g. for 45
minutes, the reaction mixture is diluted with 2.0 mM sodium
phosphate buffer (pH 7.0, ca 150 volume percent) and the THF is
removed by evacuation. The resulting suspension is filtered and the
filtrate may be purified by preparative HPLC (as described herein).
Fraction are lyophilized to isolate the conjugates. The foregoing
method is equally applicable for preparing other tubulysin
conjugates by the appropriate selection of the tubulysin starting
compound.
##STR00162##
[0305] COMPARATIVE VINBLASTINE EXAMPLE. EC145 lacking a hydrophilic
spacer linker. Peptidyl fragment Pte-Glu-Asp-Arg-Asp-Asp-Cys-OH
(Example 13) in THF was treated with either the
thiosulfonate-activated vinblastine or vinblastine pyridinyl
disulfide as a yellow solution resulting from dissolution in 0.1 M
NaHCO.sub.3 at pH >6.5 under argon. Lyophilization and HPLC gave
a 70% yield; selected 1H NMR (D.sub.2O) .delta. 8.67 (s, 1H, FA
H-7), 7.50 (br s, 1H, VLB H-11'), 7.30-7.40 (br s, 1H, VLB H-14'),
7.35 (d, 2H, J=7.8 Hz, FA H-12 & 16), 7.25 (m, 1H, VLB H-13'),
7.05 (br s, 1H, VLB H-12'), 6.51 (d, 2H, J=8.7 Hz, FA H-13 &
15), 6.4 (s, 2H, VLB H-14 & 17), 5.7 (m, 1H, VLB olefin), 5.65
(m, 1H, VLB H-7), 5.5 (d, 1H, VLB olefin), 5.5 (m, 1H, VLB H-6),
4.15 (m, 1H, VLB H-8'), 3.82 (s, 3H, VLB
C.sub.18'--CO.sub.2CH.sub.3), 3.69 (s, 3H, VLB
C.sub.16--OCH.sub.3), 2.8 (s, 3H, VLB N--CH.sub.3), 1.35 (br s, 1H,
VLB H-3'), 1.15 (m, 1H, VLB H-2'), 0.9 (t, 3H, J=7 Hz, VLB H-21'),
0.55 (t, 3H, J=6.9 Hz, VLB H-21); LCMS (ESI, m+H+) 1918.
##STR00163##
[0306] EXAMPLE. EC0234 (Mono-Saccharo-Folate Vinblastine Conjugate)
including a hydrophilic spacer linker. In a polypropylene
centrifuge bottle, folate linker (EC0233, 22 mg, 0.030 mmol) was
dissolved in 2 mL of water and bubbled with argon for 10 min. In
another flask, a 0.1N NaHCO.sub.3 solution was argon bubbled for 10
min. pH of the linker solution was carefully adjusted to 6.9 using
the 0.1N NaHCO.sub.3 solution. The vinblastine pyridinyl disulfide
(27 mg, 0.028 mmol) in 2 mL of tetrahydrofuran (THF) was added
slowly to the above solution. The resulting clear solution was
stirred under argon for 15 min to 1 h. Progress of the reaction was
monitored by analytical HPLC (10 mM ammonium acetate, pH=7.0 and
acetonitrile). THF was removed under reduced pressure and the
aqueous solution was filtered and injected on a prep-HPLC column
(X-terra Column C.sub.18, 19.times.300 mM). Elution with 1 mM
sodium phosphate pH=7.0 and acetonitrile resulted in pure fractions
containing the product. Vinblastine-saccharo-folate conjugate
(EC0234) was isolated after freeze-drying for 48 h (34 mg, 76%).
.sup.1H NMR data was in accordance with the folate conjugate.
C.sub.74H.sub.93N.sub.15O.sub.21S.sub.2; MW 1592.75; Exact Mass:
1591.61.
##STR00164##
[0307] EXAMPLE. EC0246 (Bis-Saccharo-Folate Vinblastine Conjugate).
In a polypropylene centrifuge bottle, folate linker (EC0244, 30 mg,
0.030 mmol) was dissolved in 5 mL of water and bubbled with argon
for 10 min. In another flask, a 0.1N NaHCO.sub.3 solution was argon
bubbled for 10 min, pH of the linker solution was carefully
adjusted to 6.9 using the 0.1N NaHCO.sub.3 solution. The
vinblastine pyridinyl disulfide (27 mg, 0.028 mmol) in 5 mL of
tetrahydrofuran (THF) was added slowly to the above solution. The
resulting clear solution was stirred under argon for 15 min to 1 h.
Progress of the reaction was monitored by analytical HPLC (10 mM
ammonium acetate, pH=7.0 and acetonitrile). THF was removed under
reduced pressure and the aqueous solution was filtered and injected
on a prep-HPLC column (X-terra Column C.sub.18, 19.times.300 mM).
Elution with 1 mM sodium phosphate pH=7.0 and acetonitrile resulted
in pure fractions containing the product.
Vinblastine-bis-saccbaro-folate conjugate (EC0246) was isolated
after freeze-drying for 48 h (34 mg, 66%). H NMR data was in
accordance with the folate conjugate.
C.sub.84H.sub.109N.sub.17O.sub.29S.sub.2; MW 1884.99; Exact Mass:
1883.70.
##STR00165##
[0308] EXAMPLE. EC0258 (Tris-Saccharo-Asp-Folate Vinblastine
Conjugate). In a polypropylene centrifuge bottle, folate linker
(EC0257, 37 mg, 0.031 mmol) was dissolved in 5 mL of water and
bubbled with argon for 10 min. In another flask, a 0.1N NaHCO.sub.3
solution was argon bubbled for 10 min, pH of the linker solution
was carefully adjusted to 6.9 using the 0.1N NaHCO.sub.3 solution.
The vinblastine pyridinyl disulfide (27.5 mg, 0.028 mmol) in 5 mL
of tetrahydrofuran (THF) was added slowly to the above solution.
The resulting clear solution was stirred under argon for 15 min to
1 h. Progress of the reaction was monitored by analytical HPLC (10
mM ammonium acetate, pH=7.0 and acetonitrile). THF was removed
under reduced pressure and the aqueous solution was filtered and
injected on a prep-HPLC column (X-terra Column C.sub.18,
19.times.300 mM). Elution with 1 mM sodium phosphate pH=7.0 and
acetonitrile resulted in pure fractions containing the product.
Vinblastine-tris-saccharo-Asp-folate conjugate (EC0258) was
isolated after freeze-drying for 48 h (36 mg, 62%). H NMR data was
in accordance with the folate conjugate.
C.sub.90H.sub.120N.sub.18O.sub.34S.sub.2; MW 2062.15; Exact Mass:
2060.77.
##STR00166##
[0309] EXAMPLE. EC0263 (Tris-Saccharo-Bis-Asp-Folate Vinblastine
Conjugate). In a polypropylene centrifuge bottle, folate linker
(EC0261, 37 mg, 0.029 mmol) was dissolved in 5 mL of water and
bubbled with argon for 10 min. In another flask, a 0.1N NaHCO.sub.3
solution was argon bubbled for 10 min. pH of the linker solution
was carefully adjusted to 6.9 using the 0.1N NaHCO.sub.3 solution.
The vinblastine pyridinyl disulfide (25.5 mg, 0.026 mmol) in 5 mL
of tetrahydrofuran (THF) was added slowly to the above solution.
The resulting clear solution was stirred under argon for 15 min to
1 h. Progress of the reaction was monitored by analytical HPLC (10
mM ammonium acetate, pH=7.0 and acetonitrile). THF was removed
under reduced pressure and the aqueous solution was filtered and
injected on a prep-HPLC column (X-terra Column C.sub.18,
19.times.300 mM). Elution with 1 mM sodium phosphate pH=7.0 and
acetonitrile resulted in pure fractions containing the product.
Vinblastine-tris-saccharo-bis-Asp-folate conjugate (EC0263) was
isolated after freeze-drying for 48 h (36 mg, 64%). H NMR data was
in accordance with the folate conjugate.
C.sub.94H.sub.125N.sub.19O.sub.37S.sub.2; MW 2177.24; Exact Mass:
2175.79.
##STR00167##
[0310] EXAMPLE. EC0434 (Tetra-Saccharo-Tris-Asp-Folate Vinblastine
Conjugate). In a polypropylene centrifuge bottle, folate linker
(EC0268, 20 mg, 0.012 mmol) was dissolved in 3 mL of water and
bubbled with argon for 10 min. In another flask, a 0.1N NaHCO.sub.3
solution was argon bubbled for 10 min. pH of the linker solution
was carefully adjusted to 6.9 using the 0.1N NaHCO.sub.3 solution.
The vinblastine pyridinyl disulfide (12 mg, 0.012 mmol) in 3 mL of
tetrahydrofuran (THF) was added slowly to the above solution. The
resulting clear solution was stirred under argon for 15 min to 1 h.
Progress of the reaction was monitored by analytical HPLC (10 mM
ammonium acetate, pH=7.0 and acetonitrile). THF was removed under
reduced pressure and the aqueous solution was filtered and injected
on a prep-HPLC column (X-terra Column C.sub.18, 19.times.300 mM).
Elution with 1 mM sodium phosphate pH=7.0 and acetonitrile resulted
in pure fractions containing the product.
Vinblastine-tetra-saccharo-tris-Asp-folate conjugate (EC0434) was
isolated after freeze-drying for 48 h (26 mg, 62%). .sup.1H NMR
data was in accordance with the folate conjugate.
C.sub.104H.sub.141N.sub.21O.sub.45S.sub.2; MW 2469.48; Exact Mass:
2467.88.
##STR00168##
[0311] EXAMPLE. EC0454 (Tetra-Saccharo-Bis-Asp-Folate Vinblastine
Conjugate). In a polypropylene centrifuge bottle, folate linker
(EC0452, 34 mg, 0.02 mmol) was dissolved in 3 mL of water and
bubbled with argon for 10 min. In another flask, a 0.1N NaHCO.sub.3
solution was argon bubbled for 10 min. pH of the linker solution
was carefully adjusted to 6.9 using the 0.1N NaHCO.sub.3 solution.
The vinblastine pyridinyl disulfide (20 mg, 0.02 mmol) in 3 mL of
tetrahydrofuran (THF) was added slowly to the above solution. The
resulting clear solution was stirred under argon for 15 min to 1 h.
Progress of the reaction was monitored by analytical HPLC (10 mM
ammonium acetate, pH=7.0 and acetonitrile). THF was removed under
reduced pressure and the aqueous solution was filtered and injected
on a prep-HPLC column (X-terra Column C.sub.18, 19.times.300 mM).
Elution with 1 mM sodium phosphate pH=7.0 and acetonitrile resulted
in pure fractions containing the product.
Vinblastine-tetra-saccharo-bis-Asp-folate conjugate (EC0454) was
isolated after freeze-drying for 48 h (35 mg, 70%). .sup.1H NMR
data was in accordance with the folate conjugate.
C.sub.108H.sub.151N.sub.23O.sub.43S.sub.2; MW 2523.62; Exact Mass:
2521.98.
##STR00169##
[0312] EXAMPLE. EC0455 (Tetra-Saccharo-bis-Asp-Folate Vinblastine
Conjugate). In a polypropylene centrifuge bottle, folate linker
(EC0457, 20 mg, 0.013 mmol) was dissolved in 1.5 mL of water and
bubbled with argon for 10 min. In another flask, a 0.1N NaHCO.sub.3
solution was argon bubbled for 10 min. pH of the linker solution
was carefully adjusted to 6.9 using the 0.1N NaHCO.sub.3 solution.
The vinblastine pyridinyl disulfide (18 mg, 0.018 mmol) in 1.5 mL
of tetrahydrofuran (THF) was added slowly to the above solution.
The resulting clear solution was stirred under argon for 30 min.
Progress of the reaction was monitored by analytical HPLC (10 mM
ammonium acetate, pH=7.0 and acetonitrile). THF was removed under
reduced pressure and the aqueous solution was filtered and injected
on a prep-HPLC column (X-terra Column C.sub.18, 19.times.300 mM).
Elution with 1 mM sodium phosphate pH=7.0 and acetonitrile resulted
in pure fractions containing the product.
Vinblastine-tetra-saccharo-bis-Asp-folate conjugate (EC0455) was
isolated after freeze-drying for 48 h (19 mg, 62%). .sup.1H NMR
data was in accordance with the folate conjugate.
C.sub.100H.sub.136N.sub.20O.sub.42S.sub.2; MW 2354.39.
##STR00170##
[0313] EXAMPLE. EC0456. In a polypropylene centrifuge bottle,
folate linker (EC0453, 46 mg, 0.029 mmol) was dissolved in 3 mL of
water, which had been bubbled with argon for 10 min. In another
flask, a saturated NaHCO.sub.3 solution was argon bubbled for 10
min. The pH of the linker solution was carefully adjusted, with
argon bubbling, to 6.9 using the NaHCO.sub.3 solution. The
vinblastine pyridinyl disulfide (32 mg, 1.1 eq) in 3 mL of
tetrahydrofuran (THF) was added quickly to the above solution. The
resulting clear solution was stirred under argon. Progress of the
reaction was monitored by analytical HPLC (2 mM phosphate buffer,
pH=7.0 and acetonitrile). After 30 min, to the reaction was added
12 mL 2 mM phosphate buffer (pH 7), the resulting cloudy solution
filtered, and the filtrate was injected on a prep-HPLC: Column:
Waters Xterra Prep MS Cis 10 .mu.m 19.times.250 mm; Solvent A: 2 mM
sodium phosphate, pH 7; Solvent B: ACN; Method: 5 min 1% B to 40
min 80% B 25 mL/min. Fractions containing EC0456 were collected and
freeze-dried to afford 41.6 mg fluffy yellow solid, consisting of
30 mg EC0456 (42% yield) and 11.6 sodium phosphate salt. .sup.1H
NMR and LC/MS were consistent with the product.
C.sub.104H.sub.141N.sub.21O.sub.45S.sub.2; MW 2469.48; Exact Mass:
2467.88. C, 50.58; H, 5.76; N, 11.91; 0, 29.15; S, 2.60.
##STR00171##
[0314] EXAMPLE. EC0481. In a polypropylene centrifuge bottle,
folate linker (EC0479, 12 mg, 0.0058 mmol) was dissolved in 2.5 mL
of water, which had been bubbled with argon for 10 min. In another
flask, a saturated NaHCO.sub.3 solution was argon bubbled for 10
min. The pH of the linker solution was carefully adjusted, with
argon bubbling, to 6.9 using the NaHCO.sub.3 solution. The
vinblastine pyridinyl disulfide (5.7 mg, 1.0 eq) in 2.5 mL of
tetrahydrofuran (THF) was added quickly to the above solution. The
resulting clear solution was stirred under argon. Progress of the
reaction was monitored by analytical HPLC (2 mM phosphate buffer,
pH=7.0 and acetonitrile). After 20 min, to the reaction was added
12 mL 2 mM phosphate buffer (pH 7), the resulting cloudy solution
filtered, and the filtrate was injected on a prep-HPLC: Column:
Waters Atlantis Prep T3 10 .mu.m OBD 19.times.250 mm; Solvent A: 2
mM sodium phosphate, pH 7; Solvent B: ACN; Method: 5 min 1% B to 25
min 50% B 26 mL/min. Fractions containing EC0481 were collected and
freeze-dried to afford 15.5 mg fluffy yellow solid, consisting of
10.5 mg EC0481 (60% yield) and 5.0 sodium phosphate salt. .sup.1H
NMR and LC/MS were consistent with the product. MW 2999.15; Exact
Mass: 2997.24.
##STR00172##
[0315] EXAMPLE. EC0484 (Tetra-Saccharo-Bis-.alpha.-Glu-Arg-Folate
Vinblastine Conjugate). In a polypropylene centrifuge bottle,
folate linker (EC0480, 15 mg, 0.009 mmol) was dissolved in 3 mL of
water and bubbled with argon for 10 min. In another flask, a 0.1N
NaHCO.sub.3 solution was argon bubbled for 10 min. pH of the linker
solution was carefully adjusted to 6.9 using the 0.1N NaHCO.sub.3
solution. The vinblastine pyridinyl disulfide (8.8 mg, 0.009 mmol)
in 3 mL of tetrahydrofuran (THF) was added slowly to the above
solution. The resulting clear solution was stirred under argon for
15 min to 1 h. Progress of the reaction was monitored by analytical
HPLC (10 mM ammonium acetate, pH=7.0 and acetonitrile). THF was
removed under reduced pressure and the aqueous solution was
filtered and injected on a prep-HPLC column (X-terra Column
C.sub.18, 19.times.300 mM). Elution with 1 mM sodium phosphate
pH=7.0 and acetonitrile resulted in pure fractions containing the
product. Vinblastine-tetra-saccharo-bis-.alpha.-Glu-Arg-folate
conjugate (EC0484) was isolated after freeze-drying for 48 h (16
mg, 70%). .sup.1H NMR data was in accordance with the folate
conjugate. C.sub.108H.sub.152N.sub.24O.sub.43S.sub.2; MW 2538.63;
Exact Mass: 2536.99.
##STR00173##
[0316] EXAMPLE. EC0487 (Tetra-Saccharo-Asp-Folate Vinblastine
Conjugate). In a polypropylene centrifuge bottle, folate linker
(EC0463, 21 mg, 0.015 mmol) was dissolved in 3 mL of water and
bubbled with argon for 10 min. In another flask, a 0.1N NaHCO.sub.3
solution was argon bubbled for 10 min. pH of the linker solution
was carefully adjusted to 6.9 using the 0.1N NaHCO.sub.3 solution.
The vinblastine pyridinyl disulfide (15 mg, 0.015 mmol) in 3 mL of
tetrahydrofuran (THF) was added slowly to the above solution. The
resulting clear solution was stirred under argon for 15 min to 1 h.
Progress of the reaction was monitored by analytical HPLC (10 mM
ammonium acetate, pH=7.0 and acetonitrile). THF was removed under
reduced pressure and the aqueous solution was filtered and injected
on a prep-HPLC column (Atlantis Column, 19.times.300 mM). Elution
with 1 mM sodium phosphate pH=7.0 and acetonitrile resulted in pure
fractions containing the product.
Vinblastine-tetra-saccharo-Asp-folate conjugate (EC0487) was
isolated after freeze-drying for 48 h (28 mg, 84%). .sup.1H NMR
data was in accordance with the folate conjugate.
C.sub.96H.sub.131N.sub.19O.sub.39S.sub.2; MW 2239.30; Exact Mass:
2237.83
##STR00174##
[0317] EXAMPLE. EC0489. In a polypropylene centrifuge bottle,
folate linker (EC0488, 26 mg, 0.015 mmol) was dissolved in 2.5 mL
of water, which had been bubbled with argon for 10 min. In another
flask, a saturated NaHCO.sub.3 solution was argon bubbled for 10
min. The pH of the linker solution was carefully adjusted, with
argon bubbling, to 6.9 using the NaHCO.sub.3 solution. The
vinblastine pyridinyl disulfide (15 mg, 1.0 eq) in 2.5 mL of
tetrahydrofuran (THF) was added quickly to the above solution. The
resulting clear solution was stirred under argon. Progress of the
reaction was monitored by analytical HPLC (2 mM phosphate buffer,
pH=7.0 and acetonitrile). After 20 min, to the reaction was added
12 mL 2 mM phosphate buffer (pH 7), the resulting cloudy solution
filtered, and the filtrate was injected on a prep-HPLC: Column:
Waters Xterra Prep MS C.sub.18 10 .mu.m 19.times.250 mm; Solvent A:
2 mM sodium phosphate, pH 7; Solvent B: ACN; Method: 5 min 1% B to
25 min 50% B 26 mL/min. Fractions containing EC0489 were collected
and freeze-dried to afford 35 mg fluffy yellow solid, consisting of
27.5 mg EC0489 (71% yield) and 7.5 sodium phosphate salt. .sup.1H
NMR and LC/MS were consistent with the product. MW 2550.68; Exact
Mass: 2549.01.
##STR00175##
[0318] EXAMPLE. EC0490 (Tetra-HomoSaccharo-Tris-.alpha.Glu-Folate
Vinblastine Conjugate). In a polypropylene centrifuge bottle,
folate linker (EC0478, 22 mg, 0.013 mmol) was dissolved in 3 mL of
water and bubbled with argon for 10 min. In another flask, a 0.1N
NaHCO.sub.3 solution was argon bubbled for 10 min. pH of the linker
solution was carefully adjusted to 6.9 using the 0.1N NaHCO.sub.3
solution. The vinblastine pyridinyl disulfide (mg, mmol) in 3 mL of
tetrahydrofuran (THF) was added slowly to the above solution. The
resulting clear solution was stirred under argon for 15 min to 1 h.
Progress of the reaction was monitored by analytical HPLC (10 mM
ammonium acetate, pH=7.0 and acetonitrile). THF was removed under
reduced pressure and the aqueous solution was filtered and injected
on a prep-HPLC column (X-terra Column C.sub.18, 19.times.300 mM).
Elution with 1 mM sodium phosphate pH=7.0 and acetonitrile resulted
in pure fractions containing the product.
Vinblastine-tetra-Homosaccharo-tris-Glu-folate conjugate (EC0490)
was isolated after freeze-drying for 48 h (15 mg, 45%). H NMR data
was in accordance with the folate conjugate.
C.sub.111H.sub.155N.sub.21O.sub.45S.sub.2; MW 2567.66; Exact Mass:
2565.99.
##STR00176##
[0319] EXAMPLE. EC0492 (Tetra-HomoSaccharo-Tris-.alpha.Glu-Folate
Vinblastine Conjugate). In a polypropylene centrifuge bottle,
folate linker (EC0491, 26 mg, 0.013 mmol) was dissolved in 3 mL of
water and bubbled with argon for 10 min. In another flask, a 0.1N
NaHCO.sub.3 solution was argon bubbled for 10 min. pH of the linker
solution was carefully adjusted to 6.9 using the 0.1N NaHCO.sub.3
solution. The vinblastine pyridinyl disulfide (13 mg, 0.013 mmol)
in 3 mL of tetrahydrofuran (THF) was added to the above solution.
The resulting clear solution was stirred under argon for 15 min to
1 h. Progress of the reaction was monitored by analytical HPLC (10
mM ammonium acetate, pH=7.0 and acetonitrile). THF was removed
under reduced pressure and the aqueous solution was filtered and
injected on a prep-HPLC column (X-terra Column C.sub.18,
19.times.300 mM). Elution with 1 mM sodium phosphate pH=7.0 and
acetonitrile resulted in pure fractions containing the product.
Vinblastine-tetra-homosaccharo-tris-Glu-folate conjugate (EC0492)
was isolated after freeze-drying for 48 h (22 mg, 60%). .sup.1H NMR
data was in accordance with the folate conjugate.
C.sub.122H.sub.176N.sub.24O.sub.50S.sub.2; MW 2842.97; Exact Mass:
2841.14.
##STR00177##
[0320] EXAMPLE. EC0493 (Tetra-Saccharo-tris-Glu-Folate Vinblastine
Conjugate). In a polypropylene centrifuge bottle, folate linker
(EC0477, 25 mg, 0.015 mmol) was dissolved in 1.5 mL of water and
bubbled with argon for 10 min. In another flask, a 0.1N NaHCO.sub.3
solution was argon bubbled for 10 min. pH of the linker solution
was carefully adjusted to 6.9 using the 0.1N NaHCO.sub.3 solution.
The vinblastine pyridinyl disulfide (20 mg, 0.020 mmol) in 1.5 mL
of tetrahydrofuran (THF) was added slowly to the above solution.
The resulting clear solution was stirred under argon for 30 min.
Progress of the reaction was monitored by analytical HPLC (10 mM
ammonium acetate, pH=7.0 and acetonitrile). THF was removed under
reduced pressure and the aqueous solution was filtered and injected
on a prep-HPLC column (X-terra Column C.sub.18, 19.times.300 mM).
Elution with 1 mM sodium phosphate pH=7.0 and acetonitrile resulted
in pure fractions containing the product.
Vinblastine-tetra-saccharo-tris-Glu-folate conjugate (EC0493) was
isolated after freeze-drying for 48 h (23 mg, 61%). .sup.1H NMR
data was in accordance with the folate conjugate.
C.sub.107H.sub.147N.sub.21O.sub.45S.sub.2; MW 2511.56; Exact Mass:
2509.93.
##STR00178##
[0321] EXAMPLE. EC0429. This Example including an oligoamide
hydrophilic spacer represented by the
aminoethylpiperazinylacetamide of Asp-Asp-Cys, was prepared using
the processes described herein.
[0322] The following illustrative examples of glucuronide
compounds, EC0400 and EC0423, where the saccharide based group is
illustratively introduced using click chemistry, were also prepared
as described herein.
##STR00179##
[0323] The following illustrative examples of PEG-spacer compounds,
EC0367 and EC0409, were also prepared as described herein.
##STR00180##
[0324] The following illustrative examples of sulfuric acid alkyl
ester compounds, EC0418 and EC0428, where the sulfuric acid
fragment is illustratively introduced via click chemistry, were
prepared as described herein.
##STR00181##
[0325] The following illustrative examples of additional oligoamide
spacer compounds, where the oligoamide includes an EDTE derivative
were prepared as described herein.
##STR00182##
[0326] The following illustrative examples of .beta.-alkyl
glycosides of 2-deoxyhexapyranose compounds and PEG-linked
compounds may be prepared as described herein, using click
chemistry to attach the hydrophilic groups onto the spacer
linker.
##STR00183## ##STR00184##
[0327] EC0305 lacking a hydrophilic spacer linker. EC89 (86 mg) was
taken in deionized water (4.0 mL, bubbled with argon for 10 minutes
prior to use) and the pH of the suspension was adjusted by
saturated NaHCO.sub.3 (bubbled with argon for 10 minutes prior to
use) to about 6.9 (the suspension became a solution when the pH
increased). Additional deionized water was added to the solution to
make a total volume of 5.0 mL and to the aqueous solution was added
immediately a solution of EC0312 (97 mg) in THF (5.0 mL). The
reaction mixture became homogenous quickly. After stirring under
argon for 45 minutes, the reaction mixture was diluted with 2.0 mM
sodium phosphate buffer (pH 7.0, 15 mL) and the THF was removed on
a Rotavapor. The resulting suspension was filtered and the filtrate
was injected into a preparative HPLC for purification (Column:
Waters XTerra Prep MS Cis 10 .mu.m, 19.times.250 mm; Mobile phase
A: 2.0 mM sodium phosphate buffer, pH 7.0; Mobile phase B:
acetonitrile; Method: 5% B to 80% B over 25 minutes, flow rate=25
mL/min). Fractions from 10.04-11.90 minutes were collected and
lyophilized to give EC0305 as a pale yellow fluffy solid (117
mg).
##STR00185##
[0328] EXAMPLE. General Method 2 for Preparing Conjugates having a
hydrophilic spacer linker (one-pot). Illustrated with preparation
of EC0543. DIPEA (7.8 .mu.L) and isobutyl chloroformate (3.1 .mu.L)
were added with the help of a syringe in tandem into a solution of
tubulysin A (18 mg) in anhydrous EtOAc (0.50 mL) at -15.degree. C.
After stirring for 35 minutes at -15.degree. C. under argon, to the
reaction mixture was added a solution of EC0311 (5.8 mg) in
anhydrous EtOAc (0.50 mL). The cooling was removed and the reaction
mixture was stirred under argon for an additional 45 minutes,
concentrated, vacuumed, and the residue was dissolved in THF (2.0
mL). Meanwhile, EC0488 (40 mg) was dissolved in deionized water
(bubbled with argon for 10 minutes prior to use) and the pH of the
aqueous solution was adjusted to 6.9 by saturated NaHCO.sub.3.
Additional deionized water was added to the EC0488 solution to make
a total volume of 2.0 mL and to which was added immediately the THF
solution containing the activated tubulysin. The reaction mixture,
which became homogeneous quickly, was stirred under argon for 50
minutes and quenched with 2.0 mM sodium phosphate buffer (pH 7.0,
15 mL). The resulting cloudy solution was filtered and the filtrate
was injected into a preparative HPLC for purification. Column:
Waters XTerra Prep MS C.sub.18 10 .mu.m, 19.times.250 mm; Mobile
phase A: 2.0 mM sodium phosphate buffer, pH 7.0; Mobile phase B:
acetonitrile; Method: 1% B for 5 minutes, then 1% B to 60% B over
the next 30 minutes, flow rate=26 mL/min. Fractions from
20.75-24.50 minutes were collected and lyophilized to afford EC0543
as a pale yellow fluffy solid (26 mg). The foregoing method is
equally applicable for preparing other tubulysin conjugates by the
appropriate selection of the tubulysin starting compound.
[0329] The following additional illustrative examples of tubulysin
conjugates including a hydrophilic spacer linker were prepared
using the process and syntheses described herein from
tubulysins.
##STR00186## ##STR00187##
[0330] The following Examples were also prepared as described
herein.
##STR00188##
[0331] COMPARATIVE BORTEZOMIB EXAMPLES. The following Comparative
Examples of bortezomib (Velcade) conjugates (EC0522 and EC0587)
lacking a hydrophilic spacer linker were also prepared as described
herein and in US Patent Application Publication Serial No.
2005/0002942.
##STR00189##
[0332] The following Examples of bortezomib conjugates including a
hydrophilic spacer linker were also prepared as described
herein.
##STR00190##
[0333] C85H119BN20O36S2, MW 2071.91, Exact Mass: 2070.76. Without
being bound by theory, it is appreciated that in the velcade
conjugates, the boronic acid and the linker may form intermolecular
interactions with the carbohydrate side chains. Illustratively, the
boronic acid forms boronate ester complexes with one or two
hydroxyl groups. Such ester complexes may be formed with vicinal
hydroxyls as well as with 1,3-hydroxyls. It is appreciated that the
boronate ester complexes may form at the end of the carbohydrate
fragment, or in the interior of the carbohydrate fragment. It is
further appreciated that in aqueous solution, the boronate ester
complexes may be in equilibrium with the boronic acid.
##STR00191##
[0334] COMPARATIVE .alpha.-AMANTIN EXAMPLE. The following
Comparative Example of an .alpha.-amantin conjugate lacking a
hydrophilic spacer linker was also prepared as described herein and
in US Patent Application Publication Serial No. 2005/0002942.
##STR00192##
[0335] EC0323 was not competitive with folic acid, and exhibited
the same IC50 with and without excess folic acid present.
[0336] The following Examples of an .alpha.-amantin conjugate
including a hydrophilic spacer linker was also prepared as
described herein.
##STR00193##
[0337] EC0592 Conjugate of .alpha.-amanitin. C107H154N26O50S3, MW
2700.71, Exact Mass: 2698.95. EC0592 shows an IC50 of .about.3 nM,
which may be competed with excess folic acid, against KB cells in
3H-thymidine incorporation assay.
[0338] The following Examples of illustrative conjugates were
prepared as described herein.
##STR00194## ##STR00195## ##STR00196## ##STR00197##
[0339] Prepared by Huisgen cyclization of corresponding alkyne and
azidoethylcarbohydrate; 2 eq. Na ascorbate, 1 eq. CuSO4.5H2O,
THF/water (1:1); 5 eq. Na aAscorbate, 2.5 eq. CuSO4.5H2O, THF/water
(9:1); (10 mg). C81H100N18O24S2, C, 54.84; H, 5.68; N, 14.21; 0,
21.65; S, 3.62, MW 1773.90, Exact Mass: 1772.66
##STR00198## ##STR00199## ##STR00200## ##STR00201## ##STR00202##
##STR00203## ##STR00204## ##STR00205## ##STR00206## ##STR00207##
##STR00208## ##STR00209## ##STR00210## ##STR00211## ##STR00212##
##STR00213## ##STR00214## ##STR00215## ##STR00216##
##STR00217##
* * * * *