U.S. patent application number 16/155348 was filed with the patent office on 2019-04-04 for bivalent linkers and conjugates therof.
The applicant listed for this patent is Endocyte, Inc.. Invention is credited to Stephen J. Howard, Christopher P. Leamon, Apparao Satyam, Iontcho R. Vlahov.
Application Number | 20190100494 16/155348 |
Document ID | / |
Family ID | 35134133 |
Filed Date | 2019-04-04 |
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United States Patent
Application |
20190100494 |
Kind Code |
A1 |
Vlahov; Iontcho R. ; et
al. |
April 4, 2019 |
BIVALENT LINKERS AND CONJUGATES THEROF
Abstract
Bivalent linkers to be included in or for preparing vitamin,
drug, diagnostic agent, and/or imaging agent conjugates are
described.
Inventors: |
Vlahov; Iontcho R.; (West
Lafayette, IN) ; Leamon; Christopher P.; (West
Lafayette, IN) ; Satyam; Apparao; (Mumbai, IN)
; Howard; Stephen J.; (Sherman, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Endocyte, Inc. |
West Lafayette |
IN |
US |
|
|
Family ID: |
35134133 |
Appl. No.: |
16/155348 |
Filed: |
October 9, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15412604 |
Jan 23, 2017 |
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16155348 |
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14746075 |
Jun 22, 2015 |
9550734 |
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15412604 |
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13609995 |
Sep 11, 2012 |
9090563 |
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14746075 |
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11632895 |
Jan 19, 2007 |
8288557 |
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PCT/US2005/026068 |
Jul 22, 2005 |
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13609995 |
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60590580 |
Jul 23, 2004 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 49/0002 20130101;
A61K 47/551 20170801; C07D 401/12 20130101; C07D 475/04 20130101;
A61P 43/00 20180101; C07D 213/71 20130101; A61K 47/6803
20170801 |
International
Class: |
C07D 213/71 20060101
C07D213/71; C07D 475/04 20060101 C07D475/04; C07D 401/12 20060101
C07D401/12; A61K 47/68 20060101 A61K047/68; A61K 49/00 20060101
A61K049/00; A61K 47/55 20060101 A61K047/55 |
Claims
1. A compound of the formula ##STR00045## wherein n is an integer
from 1 to about 4; R.sup.a and R.sup.b 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; and X.sup.1 and
X.sup.2 are each independently selected leaving groups, where each
of said leaving groups is displaceable by an independently selected
nucleophile.
2.-6. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims the priority benefit under 35 U.S.C.
.sctn. 119(e) of U.S. provisional patent application Ser. No.
60/590,580, filed Jul. 23, 2004, the disclosure of which is
incorporated herein in its entirety by reference.
FIELD OF THE INVENTION
[0002] This invention pertains to bivalent linkers, and the
synthesis and use thereof. In particular, this invention pertains
to the synthesis and use of bivalent linkers in preparing vitamin,
drug, diagnostic agent, and/or imaging agent conjugates.
BACKGROUND
[0003] Drug, vitamin, diagnostic, and imaging agent conjugates have
been used to treat, diagnose, and evaluate various disease states
in humans and in animals. In many cases these drug, vitamin,
diagnostic, and imaging agent conjugates include intervening
linkers separating for example a targeting ligand from a drug,
diagnostic agent, or imaging agent. These linkers include a wide
variety of bivalent fragments that may be used separately or when
linked together for inclusion in conjugates to for example space a
drug, diagnostic agent, or imaging apart from other parts of the
conjugate, such as for example a vitamin or other targeting ligand.
These linkers may also be stable to the metabolic, physiological,
or biological conditions present when they are administered to
humans and animals, or alternatively these linkers may undergo
various routes of cleavage and/or fragmentation under such
conditions. There exists a continuing need for bivalent linkers
that can be generally used in drug, vitamin, diagnostic, and
imaging agent conjugates.
SUMMARY OF THE INVENTION
[0004] Illustratively, the present invention includes divalent
linkers, which are alternatively referred to as bivalent linkers,
that may be used to couple, link, bond, attach, or otherwise
associate two or more chemical entities. This coupling, linking,
attachment, or association may be used in the formation of
conjugates of such chemical entities. Those chemical entities
include targeting ligands and receptor-binding ligands, such as
vitamins. Those chemical entities also include drugs for treating
various diseases or disease states, and imaging and diagnostic
agents for diagnosing, detecting, or otherwise monitoring various
diseases or disease states.
[0005] In one embodiment, one chemical entity includes a vitamin
receptor-binding moiety, and another entity includes a drug,
imaging agent, diagnostic agent, another bivalent linker, or
another bivalent linker conjugated with a drug, imaging agent,
diagnostic agent. It is appreciated that multiple linkers may be
used between the two or more chemical entities to change the
distance between the two entities and/or to change the
physicochemical properties of the conjugates prepared
therefrom.
[0006] In another embodiment, a compound is described that includes
a first leaving group that is displaceable by a first nucleophile,
a linker region, and a second leaving group that is displaceable by
a second nucleophile, wherein the first nucleophile is a vitamin
receptor-binding moiety, and the linker region comprises one or
more bivalent linker units, which may be the same or different, and
the second nucleophile is a drug, imaging agent, diagnostic agent,
or another bivalent linker.
[0007] In another embodiment, a compound having the structure (V)
is described
##STR00001##
wherein n is an integer from 1 to about 4; R.sup.a and R.sup.b 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; and
X.sup.1 and X.sup.2 are each independently selected leaving groups.
In one aspect, each of the independently selected leaving groups
X.sup.1 and X.sup.2 are displaceable by a nucleophile, such as a
drug, a vitamin, an imaging agent, a diagnostic agent, or another
bivalent linker nucleophile, and the like.
[0008] In another embodiment, a conjugate is formed from the
compound of formula (V) by displacing one or more of the leaving
groups X.sup.1 and X.sup.2 with a nucleophile, such as a drug, a
vitamin, an imaging agent, a diagnostic agent, or another bivalent
linker nucleophile, and the like.
[0009] In another embodiment, a compound having the structure (VI)
is described
##STR00002##
wherein m is an integer from 1 to about 4; and X.sup.1 and X.sup.2
are each independently selected leaving groups. In one aspect, each
of the independently selected leaving groups X.sup.1 and X.sup.2 is
displaceable by a nucleophile, such as a drug, a vitamin, an
imaging agent, a diagnostic agent, or another bivalent linker
nucleophile, and the like.
[0010] In another embodiment, a conjugate is formed from the
compound of formula (VI) by displacing one or more of the leaving
groups X.sup.1 and X.sup.2 with a nucleophile, such as a drug, a
vitamin, an imaging agent, a diagnostic agent, or another bivalent
linker nucleophile, and the like.
[0011] In another embodiment, a compound having the structure (VII)
is described
##STR00003##
wherein m is an integer from 1 to about 4; and X.sup.1 and X.sup.2
are each independently selected leaving groups. In one aspect, each
of the independently selected leaving groups X.sup.1 and X.sup.2 is
displaceable by a nucleophile, such as a drug, a vitamin, an
imaging agent, a diagnostic agent, or another bivalent linker
nucleophile, and the like.
[0012] In another embodiment, a conjugate is formed from the
compound of formula (VII) by displacing one or more of the leaving
groups X.sup.1 and X.sup.2 with a nucleophile, such as a drug, a
vitamin, an imaging agent, a diagnostic agent, or another bivalent
linker nucleophile, and the like.
[0013] In one aspect, the conjugate has one of the following
structures (VIII)
##STR00004##
wherein n is an integer from 1 to about 4; R.sup.a and R.sup.b 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; X.sup.1
and X.sup.2 are each independently selected leaving groups; and
L.sup.1 and L.sup.2 are each independently selected bivalent
linkers.
[0014] In another embodiment, the conjugate has one of the
following structures (IX)
##STR00005##
wherein m is an integer from 1 to about 4; X.sup.1 and X.sup.2 are
each independently selected leaving groups; and L.sup.1 and L.sup.2
are each independently selected bivalent linkers.
[0015] In another embodiment, the conjugate has one of the
following structures (X)
##STR00006##
wherein m is an integer from 1 to about 4; X.sup.1 and X.sup.2 are
each independently selected leaving groups; and L.sup.1 and L.sup.2
are each independently selected bivalent linkers.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1a shows the mass spectrum of Example 1, compound
5;
[0017] FIG. 1b shows the .sup.1H NMR spectrum of Example 1,
compound 5;
[0018] FIG. 2 shows the .sup.1H NMR spectrum of Example 3, compound
10; and
[0019] FIG. 3 shows the .sup.1H NMR spectrum of Example 4, compound
12.
DETAILED DESCRIPTION
[0020] Bivalent linkers for use in conjugates or for use in
preparing conjugates are described herein. The bivalent linkers may
be used to prepare drug conjugates, imaging agent conjugates,
and/or diagnostic agent conjugates. Drug conjugates include
targeting agent conjugates, such as the vitamin receptor-binding
drug conjugates as described in U.S. Patent Application Publication
No. US-2005-0002942-A1, and other drug conjugates described in U.S.
Patent Application Publications Nos. US-2001-0031252-A1 and
US-2003-0086900-A1. Imaging agent conjugates and diagnostic agent
conjugates include those described in U.S. Patent Application
Publication No. US-2004-0033195-A1 and International Patent
Application Publication No. WO 03/097647. The disclosures of each
of the foregoing patent application publications are incorporated
herein by reference.
[0021] It is appreciated that conjugates of analogs and derivatives
of drugs, conjugates of analogs and derivatives of vitamins,
conjugates of analogs and derivatives of imaging agents, and
conjugates of analogs and derivatives of diagnostic agents may be
prepared using the bivalent linkers described herein. Further,
unless otherwise indicated, the term drug should be understood to
include analogs and derivatives thereof, the term vitamin should be
understood to include analogs and derivatives thereof, the term
imaging agent should be understood to include analogs and
derivatives thereof, and the term diagnostic agent should be
understood to include analogs and derivatives thereof.
[0022] The bivalent linkers described herein may be used as spacers
to alter the overall distance from the drug, vitamin, imaging
agent, or diagnostic agent and the moiety to which it is
conjugated. Further, the bivalent linkers described herein may be
used to alter the physicochemical properties, solubility
properties, and other properties of the drug, vitamin, imaging
agent, or diagnostic agent conjugates in which they are
included.
[0023] Illustratively, the bivalent linkers described herein may by
included in linkers used to prepare vitamin receptor-binding drug
conjugates, vitamin receptor-binding imaging agent conjugates, and
vitamin receptor-binding diagnostic agent conjugates of the
formulae (I)
V-L-D
V-L-IA
V-L-DA (I)
where V is a vitamin receptor-binding moiety, including analogs or
derivatives thereof, L is a linker, D is a drug, including analogs
or derivatives thereof, IA is an imaging agent, including analogs
or derivatives thereof, and DA is a diagnostic agent, including
analogs or derivatives thereof. Linker (L) can comprise multiple
bivalent linkers, including the bivalent linkers described
herein.
[0024] In one embodiment, the bivalent linkers described herein are
compounds of formulae (II)
##STR00007##
where n is an integer selected from 1 to about 4; R.sup.a and
R.sup.b 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 bivalent
linkers, or other parts of the conjugate.
[0025] In another embodiment, the bivalent linkers described herein
include compounds of formulae (III)
##STR00008##
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 bivalent linkers, or other parts of
the conjugate.
[0026] In another embodiment, the bivalent linkers described herein
include compounds of formulae (IV)
##STR00009##
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 bivalent linkers, or other parts of
the conjugate.
[0027] In another embodiment, the bivalent linkers described herein
include compounds of formulae (V), (VI), and (VII)
##STR00010##
wherein n and m are each independently selected integers from 1 to
about 4; R.sup.a and R.sup.b 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; and X.sup.1 and X.sup.2 are each
independently selected leaving groups that may be nucleophilically
displaced by a drug, vitamin, imaging agent, diagnostic agent,
another bivalent linker, or another part of the conjugate.
[0028] Illustratively, vitamin-drug conjugates that may be formed
from the bivalent linkers described herein include compounds of
formulae (VIII)
##STR00011##
where V, D, and n are as described herein; n is an integer from 1
to about 4; R.sup.a and R.sup.b 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; and L.sup.1 and L.sup.2 are each
independently selected bivalent linkers used to prepare the
conjugates. Similarly, it is understood that the vitamin-imaging
agent and vitamin-diagnostic agent conjugates corresponding to the
formulae (VIII) may also be formed from the bivalent linkers
described herein.
[0029] Illustratively, vitamin-drug conjugates that may be formed
from the bivalent linkers described herein include compounds of
formulae (IX)
##STR00012##
where V, D, and n are as described herein; m is an integer from 1
to about 4; and L.sup.1 and L.sup.2 are each independently selected
bivalent linkers used to complete the conjugates. Similarly, it is
understood that the vitamin-imaging agent and vitamin-diagnostic
agent conjugates corresponding to the formulae (IX) may also be
formed from the bivalent linkers described herein.
[0030] Illustratively, vitamin-drug conjugates that may be formed
from the bivalent linkers described herein include compounds of
formulae (X)
##STR00013##
where V, D, and n are as described herein; m is an integer from 1
to about 4; and L.sup.1 and L.sup.2 are each independently selected
bivalent linkers used to complete the conjugates. Similarly, it is
understood that the vitamin-imaging agent and vitamin-diagnostic
agent conjugates corresponding to the formulae (X) may also be
formed from the bivalent linkers described herein.
[0031] It is to be further understood that when any of V, L.sup.2,
and/or D is connected to the carbonyl group of the bivalent linkers
described herein, such as the bivalent linkers of formulae (VIII),
(IX), and/or (X), the connection is made through a heteroatom, such
as an oxygen, sulfur, optionally substituted nitrogen, and the
like.
[0032] In another illustrative embodiment, intermediates useful for
preparing drug, vitamin, imaging agent, or diagnostic agent
conjugates are described herein. Such intermediates may be
subsequently linked to other components to form vitamin, imaging
agent, or diagnostic agent conjugates. Intermediates described
herein include compounds of formulae XI
##STR00014##
where V, D, L.sup.1, L.sup.2, X.sup.1, and X.sup.2 are as described
herein; and n is an integer from 1 to about 4. Similarly, it is
understood that the vitamin-imaging agent and vitamin-diagnostic
agent conjugates corresponding to the formulae (XI) may also be
formed from the bivalent linkers described herein.
[0033] Illustratively, vitamin-drug conjugates that may be formed
from the bivalent linkers described herein include compounds of
formulae (XII)
##STR00015##
where V, D, L.sup.1, L.sup.2, X.sup.1, and X.sup.2 are as described
herein; and m is an integer from 1 to about 4. Similarly, it is
understood that the vitamin-imaging agent and vitamin-diagnostic
agent conjugates corresponding to the formulae (XII) may also be
formed from the bivalent linkers described herein.
[0034] Illustratively, vitamin-drug conjugates that may be formed
from the bivalent linkers described herein include compounds of
formulae (XIII)
##STR00016##
where V, D, L.sup.1, L.sup.2, X.sup.1, and X.sup.2 are as described
herein; and m is an integer from 1 to about 4. Similarly, it is
understood that the vitamin-imaging agent and vitamin-diagnostic
agent conjugates corresponding to the formulae (XIII) may also be
formed from the bivalent linkers described herein.
[0035] It is further understood that when any of V, L.sup.2, and/or
D is connected to the carbonyl group of the bivalent linkers
described herein, such as the bivalent linkers of formulae (XI),
(XII), and (XIII), the connection is made through a heteroatom,
such as an oxygen, sulfur, optionally substituted nitrogen, and the
like.
[0036] In one illustrative embodiment, the leaving group X.sup.1 is
an arylthio group. In one aspect, the arylthio group is an
heteroarylthio group and includes, but is not limited to,
optionally substituted 2-pyridinylthio, optionally substituted
4-pyridinylthio, and the like. In another illustrative aspect, the
substitutions include electron withdrawing substituents, such as
cyano, nitro, alkylsulfonyl, arylsulfonyl, halo, haloalkyl, acyl
and derivatives thereof, carboxyl and derivatives thereof, and the
like, and combinations thereof.
[0037] In another illustrative embodiment, the leaving group
X.sup.2 is an aryloxy group. In one aspect, the aryloxy group is an
optionally substituted phenyl group. In another illustrative
aspect, the aryloxy group is an optionally substituted
heteroaryloxy group and includes, but is not limited to, optionally
substituted benzotriazoles, and the like. In another illustrative
aspect, the substitutions include electron withdrawing
substituents, such as cyano, nitro, alkylsulfonyl, arylsulfonyl,
halo, haloalkyl, acyl and derivatives thereof, carboxyl and
derivatives thereof, and the like, and combinations thereof.
[0038] Illustrative leaving groups X.sup.1 include compounds of
formulae (XIV)
##STR00017##
where (*) indicates the point of attachment to the sulfur of the
bivalent linkers described herein. In one aspect, each of the
leaving groups X.sup.1 having the formulae (XIV) may be optionally
substituted with one or more substituents, or additional
substituents, selected from halo, alkyl, haloalkyl, alkoxy,
haloalkoxy, cyano, nitro, and the like. Further illustrative
leaving groups include alkyl and arylsulfonyl leaving groups, such
as but not limited to
##STR00018##
where R is alkyl or aryl, each of which may be optionally
substituted, such as with halo, alkyl, haloalkyl, alkoxy,
haloalkoxy, cyano, nitro, and the like; and where (*) indicates the
point of attachment to the sulfur of the bivalent linkers described
herein.
[0039] Illustrative leaving groups X.sup.2 include compounds of
formulae (XV)
##STR00019##
where (*) indicates the point of attachment to the carbonyl of the
bivalent linkers described herein. In one aspect, each of the
leaving groups X.sup.2 having the formulae (XV) may be optionally
substituted with one or more substituents, or additional
substituents, selected from halo, alkyl, haloalkyl, alkoxy,
haloalkoxy, cyano, nitro, and the like.
[0040] Bivalent linkers that include leaving groups such as those
shown in formulae XIII, XIV, and XV may be prepared following the
corresponding synthetic procedures described in the Examples
described herein.
[0041] In another illustrative aspect, the vitamin receptor binding
drug delivery conjugate intermediate described herein includes a
compound having the formulae:
##STR00020##
or a protected derivative thereof, where R.sup.a and R.sup.b 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; Y is
hydrogen or a substituent, illustratively an electron withdrawing
substituent, including but not limited to nitro, cyano, halo,
alkylsulfonyl, a carboxylic acid derivative, and the like; 1.sub.s
is either a bond or another bivalent linker; and where V is as
defined herein. It is appreciated that other substituents may be
optionally present on the cysteine or homocysteine portion of these
drug delivery conjugates, such as longer and/or branched alkyl
groups, alkoxy groups, alkoxyalkyl groups, and the like. It is to
be understood that cyclic variants of the cysteine portion of these
drug delivery conjugates are contemplated. In one aspect, R.sup.a
is hydrogen, and R.sup.b is alkyl, such as methyl. In another
aspect, both R.sup.a and R.sup.b are alkyl, either the same or
different, such as both being methyl. In another aspect, R.sup.a
and R.sup.b are taken together with attached carbon to form a spiro
cyclopropyl.
[0042] In another illustrative aspect of the vitamin receptor
binding drug delivery conjugate intermediate described herein, the
intermediate includes compounds having the formulae:
##STR00021##
or protected derivatives thereof, where V is the vitamin, or an
analog or a derivative thereof, AA is an amino acid, illustratively
selected from the naturally occurring amino acids, or stereoisomers
thereof, R.sup.a and R.sup.b 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, Y is hydrogen or a substituent,
illustratively an electron withdrawing substituent, including but
not limited to nitro, cyano, halo, alkylsulfonyl, a carboxylic acid
derivative, and the like, n and m are independently selected
integers, such as 1, 2, or 3, and p is an integer such as 1, 2, 3,
4, or 5. It is appreciated that other substituents may be
optionally present on the cysteine or homocysteine portion of these
drug delivery conjugates, such as longer and/or branched alkyl
groups, alkoxy groups, alkoxyalkyl groups, and the like. It is to
be understood that cyclic variants of the cysteine portion of these
drug delivery conjugates are contemplated. AA can 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 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.
[0043] It is appreciated that the bivalent linkers described herein
may undergo cleavage under certain chemical, environmental, or
physiological conditions. In particular, the bivalent linkers
described herein may undergo cleavage under physiological
conditions, such as by the action of a glutathione mediated
mechanism. In such embodiments, those linkers may be alternatively
referred to as releasable linkers.
[0044] Illustrative mechanisms for cleavage of the bivalant linkers
described herein include the following 1,4 and 1,6 fragmentation
mechanisms
##STR00022##
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 bivalent 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 bivalent 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 bivalent 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 releaseable nature of the
illustrative bivalent linkers described herein may be realized by
whatever mechanism may be relevant to the chemical, metabolic,
physiological, or biological conditions present.
General Disulfide Formation
[0045] Disulfide groups can be generally formed by reacting an
alkyl or aryl sulfonylthioalkyl derivative, or the corresponding
heteroaryldithioalkyl derivative such as a pyridin-2-yldithioalkyl
derivative, and the like, with an alkylenethiol derivative, as
illustrated in Scheme 1.
##STR00023##
Solvents that can be used for this reaction include THF, EtOAc,
CH.sub.2Cl.sub.2, CHCl.sub.3, CCl.sub.4, DMF, DMSO, and the like.
The temperature range employed in this transformation may vary
between 0.degree. C. and 80.degree. C. The required alkyl or aryl
sulfonylthioalkyl derivative may be prepared using art-recognized
protocols, and also according to the method of Ranasinghe and
Fuchs, Synth. Commun. 18(3), 227-32 (1988), the disclosure of which
is incorporated herein by reference. Other methods of preparing
unsymmetrical dialkyl disulfides are based on a transthiolation of
unsymmetrical heteroaryl-alkyl disulfides, such as 2-thiopyridinyl,
3-nitro-2-thiopyridinyl, and like disulfides, with alkyl thiol, as
described in WO 88/01622, European Patent Application No.
0116208A1, and U.S. Pat. No. 4,691,024, the disclosures of which
are incorporated herein by reference.
General Carbonate Thiocarbonate and Carbamate Formation
[0046] Carbonates, thiocarbonates, and carbamates can generally be
formed by reacting an hydroxy-substituted compound, a
thio-substituted compound, or an amine-substituted compound,
respectively, with an activated alkoxycarbonyl derivative where X
is a suitable leaving group, as illustrated in Scheme 2.
##STR00024##
where Q is oxygen, sulfur, optionally substituted nitrogen,
optionally protected nitrogen, and the like. Solvents that can be
used for this reaction include THF, EtOAc, CH.sub.2Cl.sub.2,
CHCl.sub.3, CCl.sub.4, DMF, DMSO, and the like. The temperature
range employed in this transformation may vary between 0.degree. C.
and 80.degree. C. Any basic catalyst such as an inorganic base, an
amine base, a polymer bound base, and the like can be used to
facilitate the reaction.
EXAMPLES
Example 1:
6-Trifluoromethyl-1-[2-(2-pyridinyldithio)ethoxycarbonyloxy]ben-
zotriazole
[0047] Example 1 was prepared according to Scheme 3.
##STR00025##
[0048] Step (a). A solution of 4-(dimethylamine)pyridine (3.0 g;
24.68 mmol; 1.03 eqs.) in 10 mL anhydrous methanol was added to a
suspension of 2-(2-pyridyldithio)ethanol hydrochloride (compound
(1a); C.sub.7H.sub.9NS.sub.2O.HCl; 5.4 g; 24 mmol) in 10 ml of
anhydrous methanol, and the mixture was stirred to form a clear
solution. Within a few minutes, the solution turned turbid with the
formation of a fine suspension, and this suspension was purified by
flash chromatography (FC). FC was performed by using 140 g silica
gel 60 with 5% methanol in CH.sub.2Cl.sub.2 to form a 24
cm.times.4.3 cm silica gel bed with 500 ml solvent reservoir. The
said suspension in 20 mL CH.sub.2Cl.sub.2 was loaded and the column
was eluted with 5% methanol in CH.sub.2Cl.sub.2 with standard
collection of 30 mL fractions coupled with UV-detection. The
product compound (1b) can be detected from fraction 2 to 9 by TLC
(1:1 Hexane:EtOAc).
[0049] Step (b). 1-Hydroxy-6-(trifluoromethyl)benzotriazole
(compound (2); C.sub.7H.sub.4F.sub.3N.sub.3O; 2.3 g; 0.11 mol;
Aldrich) was dissolved in 770 mL ether and the supernatant was
decanted into a 2 L RB flask Trichloromethyl chloroformate
(compound (3); C.sub.2Cl.sub.4O.sub.2; 107 g; 0.053 mol; 6.0 mL)
was added into this clean colorless solution at room temperature
over a 15 minutes period. The mixture was gently heated to
temperature between 40-50.degree. C. for 1 hour, and cooled to room
temperature. The solution was filtered, and the precipitate was
washed with ether (10.times.30 mL). The precipitate was a white
powder and was dried with vacuum and gave 16.95 g (74% yield).
Additional synthetic details are described by Ogura et al, in
Synthesis, 1987, 557-560, the disclosure of which is incorporated
herein by reference.
[0050] Step (c). A solution of compound (1b) (ca. 7.5 mmol) in 10
mL acetonitrile was added over a 2 minutes period to a stirred
clean solution of compound 4 (3.46 g; 8 mmol) in 300 ml
acetonitrile at room temperature. The mixture was then stirred at
room temperature for 24 hours. TLC analysis of the mixture was
performed after 24 hours. The mixture was allowed to be stirred for
additional 14 hours to a total of 38 hours. The mixture was
concentrated and washed with 50 mL of 1N NaHCO.sub.3 eq. and 100 mL
of ethyl acetate. The organic layer was separated, washed further
with 1 N NaHCO.sub.3 eq. (1.times.10 mL), dried (Na.sub.2SO.sub.4),
and filtered, to give a white solid powder, which was dried on
vacuum for 5 hours and gave 2.54 g.
Example 2
[0051] Example 2 was prepared according to Scheme 4.
##STR00026##
[0052] 2-(2-pyridyldithio)ethanol hydrochloride (compound (1b); 8.8
g; 39.33 mL) was dissolved in 78 mL CH.sub.2Cl.sub.2, and 2 eqs. of
pyridine (C.sub.5H.sub.5N; 80.88 mL) was added. para-Nitrophenyl
chloroformate (compound (6); 8.08 g; 40 mmol) was dissolved in 80
mL CH.sub.2Cl.sub.2, and the solution was added to mixture of
2-(2-pyridyldithio)ethanol hydrochloride and pyridine over a 15
minutes period. The resultant clear solution was stirred at room
temperature for 15 hours. TLC analysis for the above mixture after
15 hours stirring indicated completion of the reaction. The mixture
was then filtered to remove precipitated pyridine hydrochloride.
The light yellow clear filtrate was washed with deionized water
(2.times.50 mL) to remove the dissolved pyridine hydrochloride,
dried (Na.sub.2SO.sub.4), filtered, and concentrated with vacuum
(13.8 g). Silica gel 60 (250 g) in CH.sub.2Cl.sub.2 was used to
from a 50 cm.times.4.2 cm of silica gel bed with 250 mL solvent
reservoir. The product compound (7), ca. 13.8 g, was dissolved in
15 mL (10 mL+5 mL) of CH.sub.2Cl.sub.2 and the solution was loaded
with an elution ratio of 30 mL/min with standard collection of 30
mL fractions coupled with UV-detection and gave 13.3 g (96%
yield).
Example 3
[0053] Example 3 was prepared according to Scheme 5.
##STR00027##
[0054] 3-Nitro-2-pyridinesulfenyl chloride (compound (8)) was
dissolved in 15 mL anhydrous CH.sub.2Cl.sub.2. The solution was
cooled by an ice bath. 4-ethanoyl thiophenol (compound (9)) was
dissolved in 10 mL CH.sub.2Cl.sub.2 and added to a dropping funnel
affixed to the container containing 3-nitro-2-pyridinesulfenyl
chloride solution. The solution of 4-ethanoyl thiophenol was added
over a 2 to 5 minute period. After the solution of 4-ethanoyl
thiophenol was added, the mixture was allowed to warm to room
temperature and then stirred for 2 hours. While addition of
4-ethanoyl thiophenol, a precipitate formed. After the 2 hours
period of stirring at room temperature was completed, the mixture
was sonicated for 5 minutes and the precipitate dissolved. TLC
analysis showed a new spot formed in addition to
3-nitro-2-pyridinesulfenyl chloride and 4-ethanoyl thiophenol. The
reaction mixture was washed with saturated NaHCO.sub.3. The organic
layer was supplemented with an addition 100 mL Ch.sub.2Cl.sub.2.
The product was dried for 3 hours under vacuum.
Example 4
[0055] Example 4 was prepared according to Scheme 6.
##STR00028##
[0056] Compound (10) (0.025 g; 0.085 mmol) was dissolved in
anhydrous CH.sub.2Cl.sub.2 under stirring. para-Nitrophenyl
cholorofomate (compound (11); 0.020 g; 1.2 eq.) was added along
with 1 eq. of triethylamine (TEA) to the stirring solution. TLC
analysis showed that compound (10) was consumed after 2 hours. The
product compound (12) was isolated by column chromatography (7:3
Hexane:EtOAc).
Example 5: General Preparation of Compounds Containing a Cysteine
Disulfide Bond
[0057] Any of thiosulfonates (13) (1 eq.), prepared according to
the method of Ranasinghe and Fuchs, Synth. Commun. 18(3), 227-32
(1988), the disclosure of which is incorporated herein by
reference, are reacted with drugs, drug analogs, or drug
derivatives (14) (1 eq.) to prepare the drug thiosulfonates (15) as
a solution in methanol, as shown in Scheme 7.
##STR00029##
Referring to Scheme 7, R is alkyl or aryl, L is a suitable leaving
group, such as halogen, pentafluorophenyl, and the like, n is an
integer from 1 to about 4, and X is --O--, --NH--, --C(O)O--, or
--C(O)NH--. Conversion is conveniently monitored by observing the
disappearance of each starting material by TLC (silica gel;
CHCl.sub.3/MeOH=9/1). Final yield was 83% (mass of removed product
was 32 mg from a total yield of 38.9 mg).
[0058] The folate-containing peptidyl fragment
Pte-Glu-(AA).sub.n-Cys-OH (18) is prepared by a polymer-supported
sequential approach using the Fmoc-strategy on an acid-sensitive
Fmoc-Cys(Trt)-Wang resin (16), as shown in Scheme 8.
##STR00030##
[0059] Referring to Scheme 8, R.sub.1 is Fmoc, R.sub.2 is Trityl,
and DIPEA is diisopropylethylamine. PyBop is applied as the
activating reagent to ensure efficient coupling. Fmoc protecting
groups are removed after each coupling step under standard
conditions. 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 8, and represented by in step (b) by
Fmoc-AA-OH. Thus, AA refers to any amino acid starting material,
that is appropriatedly protected.
[0060] The coupling sequence (steps (a) & (b)) involving
Fmoc-AA-OH is performed "n" times to prepare solid-supported
peptide (17), where n is an integer and may equal 0 to about 100.
Following the last coupling step, the remaining Fmoc group is
removed, 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. The TFA protecting group is removed upon
treatment with base (step (f)) to provide the folate-containing
Cys-containing peptidyl fragment (18).
[0061] Drug conjugates are prepared by reacting folate derivative
(18) (0.9-0.95 eq.) with drug thiosulfonate (15) in deionized water
(0.04 M, pH adjusted to 7 with 0.1 N NaHCO.sub.3) under argon for
about 30 minutes, forming a disulfide bond. Upon evaporation of the
methanol in vacuo, the conjugate may be purified by preparative
HPLC (Prep Novapak HR C18 19.times.300 mM column; mobile phase
(A)-1.0 mM phosphate buffer, pH=6; organic phase (B)-acetonitrile;
conditions-gradient from 99% A and 1% B to 50% A and 50% B in 30
minutes, flow rate=15 mL/minute).
Examples 6a-6f
[0062] Examples 6a-6f were prepared by the following general
procedure. To a well stirred solution of the corresponding drug
having an --OH group (1 eq. in dry CH.sub.2Cl.sub.2 or dry THF) was
added under argon 6-(trifluoromethyl)benzotriazolyl
2-(2'-pyridyldithioethyl carbonate (1.3 eq.) and
NN-dimethylaminopyridine (1.5 eq.). The reaction mixture was
stirred for 3 h, and the pyridyldithio-derivatized drug was
isolated by silica chromatography (>65% for each example). The
corresponding peptidyl fragment (0.5 eq.), prepared according to
the procedures described herein and alternatively by conventional
procedures, such as those described in U.S. Patent Application
Publication No. US-2005-0002942-A1, was dissolved in DMSO. To the
resulting clear yellow solution was added the pyridyl-dithio
derivatized drug. After 30 min, the reaction was completed and the
conjugate purified by HPLC. In the case of Example 6e, the peptidyl
fragment Pte-Glu-Asp-Arg-Asp-Asp-Cys-OH was first dissolved in
water, and the pH of the solution was adjusted to 2.5 with 0.1 N
HCl, causing the peptidyl fragment to precipitate. The peptidyl
fragment was collected by centrifugation, dried, and dissolved in
DMSO for subsequent reaction with the pyridyldithio-derivatized
drug.
Example 6a
##STR00031##
[0064] .sup.1H NMR (DMSO-d.sub.6) .delta. 4.7 (d, 1H), 4.95 (t,
1H), 6.7 (d, 4H), 6.9 (t, 1H), 7.95 (d, 2H), 8.1 (d, 2H), 8.2 (m,
1H), 8.3 (s, 1H), 8.4 (s, 1H), 8.7 (s, 1H), 10.2 (s, 1H), 11.8 (d,
2H).
Example 6b
##STR00032##
[0066] ES MS (m-H).sup.- 1436.4, (m+H).sup.+ 1438.3.
Example 6c
##STR00033##
[0068] .sup.1H NMR (DMSO-d.sub.6/D.sub.2O) .delta. 1.0 (s, 1H), 1.1
(s, 1H), 1.6 (s, 1H), 1.8 (s, 1H), 2.1 (s, 1H), 2.25 (s, 3H), 2.65
(dd, 2H), 3.7 (d, 1H), 4.4 (t, 1H), 4.55 (q, 2H), 4.6 (d, 2H), 4.95
(d, 1H), 5.9 (t, 1H), 6.15 (s, 1H), 6.6 (d, 2H), 7.85 (d, 2H), 7.95
(d, 2H), 8.6 (s, 1H), 8.95 (d, 1H).
Example 6d
##STR00034##
[0070] .sup.1H NMR (DMSO-d.sub.6/D.sub.2O) .delta. 1.0 (s, 1H), 1.1
(s, 1H), 1.65 (s, 1H), 2.1 (s, 1H), 2.25 (s, 3H), 2.6 (dd, 2H),
3.25 (dd, 1H), 3.6 (t, 2H), 3.7 (d, 1H), 4.4 (t, 1H), 4.6 (d, 1H),
4.95 (d, 1H), 5.9 (t, 1H), 6.2 (s, 1H), 6.6 (d, 2H), 7.7 (t, 1H),
7.9 (d, 2H), 7.95 (d, 2H), 8.6 (s, 1H), 9.1 (d, 2H).
Example 6e
##STR00035##
[0072] .sup.1H NMR (DMSO-d.sub.6/D.sub.2O) .delta. 10.85 (d, 2H),
1.05 (d, 2H), 1.2 (d, 2H), 1.7 (d, 2H), 3.95 (d, 1H), 4.05 (dd,
1H), 5.4 (dd, 1H), 5.7 (dd, 1H), 6.65 (d, 2H), 7.6 (d, 2H), 7.95
(s, 1H), 8.65 (s, 1H).
Example 6f
##STR00036##
[0074] ES MS (m+H).sup.+ 1487.23; .sup.1H NMR
(DMSO-d.sub.6/D.sub.2O) .delta. 0.9 (t, 2H), 1.3 (t, 2H), 2.15 (t,
2H), 3.2 (dd, 1H), 4.0 (t, 1H), 4.15 (q, 1H), 5.3 (s, 2H), 5.5 (s,
2H), 6.6 (d, 2H), 7.0 (s, 1H), 7.4 (m, 2H), 7.55 (d, 2H), 8.0 (d,
2H), 8.6 (s, 1H).
Example 7
##STR00037##
[0076] The intermediate 4-(2-pyridinyldithio)benzylcarbonate of SN
38 (10-hydroxy-7-ethylcamptothecin) was prepared according to the
procedure described by P. Senter et al., J. Org. Chem. 1990, 55,
2875, the disclosure of which is incorporated herein by reference.
The peptidyl fragmant Pte-Glu-Asp-Arg-Asp-Cys-OH, prepared as
described herein, was dissolved in DMSO, and to the resulting clear
yellow solution was added the pyridyl-dithio derivatized drug.
After 30 min, the reaction was completed and the conjugate purified
by HPLC; ES MS (m+H).sup.+ 1425.38; .sup.1H NMR
(DMSO-d.sub.6/D.sub.2O) .delta. 0.9 (t), 1.15 (t), 3.9 (t), 4.0
(t), 4.25 (t), 5.1 (m), 5.2 (s), 5.4 (s), 6.55 (d), 7.25 (d), 7.35
(d), 7.5 (d), 7.9 (d), 8.55 (s).
Example 8
##STR00038##
[0078] The compound of Example 8 was prepared from the peptidyl
fragment Pte-Glu-Asp-Arg-Asp-Asp-Cys-OH, prepared according to the
procedures described herein and alternatively by conventional
procedures, such as those described in U.S. Patent Application
Publication No. US-2005-0002942-A1. The peptidyl fragment also
reacted with either the thiosulfonate or pyridyldithio-activated
vinblastine to form Example 8. The pyridyldithio-activated
vinblastine intermediates were prepared using the procedures
described herein for other examples.
Examples 9-13
[0079] The compounds of Examples 9-13 were prepared according to
the procedures generally described herein, and were characterized
by electrospray mass spectroscopy (ES MS), and other spectroscopic
techniques, including 1D and 2D NMR, and UV, illustrative results
of which are described herein.
Example 9
##STR00039##
[0081] UV (nm) 233 (max), 255, 280; .sup.1H NMR (D.sub.2O, NaOD,
CD.sub.3CN) .delta. 1.15 (d, 3H), 2.3 (s, 3H), 3.6 (s, 1H), 3.85
(s, 3H), 4.9 (s, 1H), 5.3 (s, 1H), 6.5 (d, 2H), 7.3 (m, 1H), 7.5
(d, 2H), 7.65 (d, 2H), 8.4 (s, 1H).
Example 10
##STR00040##
[0083] ES MS (m+H).sup.+ 1382.3, (m+Na).sup.+ 1405.4.
Example 11
##STR00041##
[0084] Example 12
##STR00042##
[0085] Example 13
##STR00043##
[0087] The foregoing exemplary embodiments are intended to be
illustrative of the invention, and should not be interpreted or
construed as limiting in any way the invention as described herein.
For example, compounds generally represented by the following
illustrative vitamin-drug conjugate are to be included in the
invention as described herein
##STR00044##
where R.sup.1 and R.sup.2 are each independently hydrogen or alkyl,
such as methyl; and 1.sub.H is a heteroatom, such as oxygen,
sulfur, optionally substituted nitrogen, or optionally protected
nitrogen, and the like.
* * * * *