U.S. patent application number 13/748648 was filed with the patent office on 2013-08-01 for integrin antagonist conjugates for targeted delivery to cells expressing lfa-1.
This patent application is currently assigned to Hoffmann-La Roche Inc.. The applicant listed for this patent is Hoffmann-La Roche Inc.. Invention is credited to Robert A. Goodnow, JR., Matthew Michael Hamilton, Agnieszka Kowalczyk, Achyutharao Sidduri.
Application Number | 20130197059 13/748648 |
Document ID | / |
Family ID | 47605526 |
Filed Date | 2013-08-01 |
United States Patent
Application |
20130197059 |
Kind Code |
A1 |
Goodnow, JR.; Robert A. ; et
al. |
August 1, 2013 |
INTEGRIN ANTAGONIST CONJUGATES FOR TARGETED DELIVERY TO CELLS
EXPRESSING LFA-1
Abstract
The invention relates to compounds of formula I: ##STR00001##
wherein R1, R2, and n are defined in the detailed description and
claims. In particular, the present invention relates to the
compounds of formula I for use in the manufacture and delivery of
conjugated moieties such as small molecules, peptides, nucleic
acids, fluorescent moieties, and polymers which are linked to LFA-1
integrin antagonists to target cells expressing LFA-1.
Inventors: |
Goodnow, JR.; Robert A.;
(Gillette, NJ) ; Hamilton; Matthew Michael;
(Hackettstown, NJ) ; Kowalczyk; Agnieszka; (Mine
Hill, NJ) ; Sidduri; Achyutharao; (West Orange,
NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hoffmann-La Roche Inc.; |
Nutley |
NJ |
US |
|
|
Assignee: |
Hoffmann-La Roche Inc.
Nutley
NJ
|
Family ID: |
47605526 |
Appl. No.: |
13/748648 |
Filed: |
January 24, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61591297 |
Jan 27, 2012 |
|
|
|
61678673 |
Aug 2, 2012 |
|
|
|
Current U.S.
Class: |
514/44A ;
514/275; 514/425; 514/513; 536/24.5; 544/331; 546/294; 548/546;
558/254 |
Current CPC
Class: |
C07C 381/00 20130101;
A61P 43/00 20180101; A61P 29/00 20180101; C09B 11/22 20130101; A61P
3/00 20180101; A61K 47/54 20170801; C07D 207/36 20130101; C07D
207/46 20130101; C07D 213/71 20130101; C07C 327/06 20130101; C07H
21/02 20130101; A61K 47/60 20170801; A61P 35/00 20180101; C07D
403/12 20130101; A61K 47/545 20170801 |
Class at
Publication: |
514/44.A ;
544/331; 514/275; 548/546; 514/425; 558/254; 514/513; 546/294;
536/24.5 |
International
Class: |
C07D 403/12 20060101
C07D403/12; C07H 21/02 20060101 C07H021/02; C07D 213/71 20060101
C07D213/71; C07D 207/46 20060101 C07D207/46; C07C 327/06 20060101
C07C327/06 |
Claims
1. A compound of formula I: ##STR00084## or a pharmaceutically
acceptable salt or ester thereof; wherein n is 1-24 and wherein: R1
is selected from the group consisting of: (1) a compound of the
formula: ##STR00085## (2) a compound of the formula: ##STR00086##
and (3) a compound of the formula: ##STR00087## wherein Q is H or
OH; R2 is selected from the group consisting of: (1) a compound of
the formula: ##STR00088## (2) a compound of the formula:
##STR00089## (3) a compound of the formula: ##STR00090## and (4) a
compound of the formula: ##STR00091## wherein R3 is a conjugated
moiety and X represents either sulfur or a compound of the formula:
##STR00092##
2. A compound of claim 1, wherein R1 is a compound of the formula:
##STR00093##
3. A compound of claim 1, wherein R1 is a compound of the formula:
##STR00094##
4. A compound of claim 1, wherein R1 is a compound of the formula:
##STR00095## wherein Q is H or OH.
5. A compound of claim 4, wherein Q is H.
6. A compound of claim 4, wherein Q is OH.
7. A compound of claim 1, wherein R2 is a compound of the formula:
##STR00096##
8. A compound of claim 1, wherein R2 is a compound of the formula:
##STR00097##
9. A compound of claim 1, wherein R2 is a compound of the formula:
##STR00098##
10. A compound of claim 1, wherein R2 is a compound of the formula:
##STR00099## wherein R3 is a single or double stranded
oligonucleotide and X represents either sulfur or a compound of the
formula: ##STR00100##
11. A compound of claim 10, wherein X represents sulfur.
12. A compound of claim 10, wherein R3 is a siRNA molecule.
13. A compound of claim 12, wherein R1 a compound of the formula:
##STR00101##
14. A compound of claim 12, wherein R1 a compound of the formula:
##STR00102##
15. A compound of claim 12, wherein R1 a compound of the formula:
##STR00103## wherein Q is H or OH.
16. A compound of claim 1, selected from the group consisting of:
(S)-3-{3-{2-[2-(2-{2-[3-(2,5-Dioxo-2,5-dihydro-pyrrol-1-yl)-propionylamin-
o]-ethoxy]-ethoxy}-ethoxy)-ethoxy]-propionylamino}-2-({2-[3-(3-hydroxy-phe-
nyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}amino)propionic
acid; and
(S)-3-{3-(2-{2-[2-(2-{2-[2-(2-{2-[3-(2,5-Dioxo-2,5-dihydro-pyrrol-1-y-
l)-propionylamino]-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-
-ethoxy]-propionylamino}-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dime-
thyl-pyrimidine-5-carbonyl}amino)propionic acid.
17. A compound of claim 1, selected from the group consisting of:
(S)-3-{3-[2-(2-{2-[2-(2-{2-[2-(2-{2-[2-(2-{2-[3-(2,5-Dioxo-2,5-dihydro-py-
rrol-1-yl)-propionylamino]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-
-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-propionylamino}-2-({2-[3--
(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}amino)p-
ropionic acid; and
(S)-3-{4-[4-(3-(2-{2-[2-(2-{2-[2-(2-{2-ethoxy)-ethoxy]-ethoxy}-ethoxy)-et-
hoxy]-ethoxy}-ethoxy)-ethoxy]-amino-propoxy)-2,6-dichloro-benzoylamino]-ph-
enyl}-2-[2-chloro-4-(3-hydroxy-benzylcarbamoyl)-benzoylamino]-propionic
acid-PEGS.
18. A compound of claim 1, selected from the group consisting of:
S)-2-[2-Chloro-4-(3-hydroxy-benzylcarbamoyl)-benzoylamino]-3-(4-{2,6-dich-
loro-4-[3-(3-{2-[2-(2-{2-[3-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-propionyla-
mino]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-propionylamino)-propoxy]-benzoylamin-
o}-phenyl)-propionic acid; and
S)-2-[2-Chloro-4-(3-hydroxy-benzylcarbamoyl)-benzoylamino]-3-(4-{2,6-dich-
loro-4-[3-(3-{2-[2-(2-{2-2-[2-(2-{2-2-[2-(2-{2-[3-(2,5-dioxo-2,5-dihydro-p-
yrrol-1-yl)-propionylamino]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy}-ethoxy-
)-ethoxy]-ethoxy}-propionylamino)-propoxy]-benzoylamino}-phenyl)-propionic
acid.
19. A compound of claim 1, selected from the group consisting of:
S)-2-[2-Chloro-4-(3-hydroxy-benzylcarbamoyl)-benzoylamino]-3-(4-{2,6-dich-
loro-4-[3-(3-{2-[2-(2-{2-2-[2-(2-{2-[3-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-
-propionylamino]-ethoxy}-ethoxy)-ethoxy]-ethoxy]-ethoxy}-ethoxy)-ethoxy]-e-
thoxy}-propionylamino)-propoxy]-benzoylamino}-phenyl)-propionic
acid; and
(S)-3-{[(3-{3-[3-(2-{2-[2-(2-{2-[2-(2-{2-[3-(2,5-Dioxo-2,5-dihydro-pyrrol-
-1-yl)-propionylamino]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-eth-
oxy}-ethoxy)-propionylamino]-propyl-oxy}-phenyl)-carbonyl]-amino}-2-({2-[3-
-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino-
)-propionic acid.
20. A compound of claim 1, selected from the group consisting of:
(S)-3-{[(3-{3-[3-(2-{2-[2-(2-{2-[2-(2-{2-[3-(2,5-Dioxo-2,5-dihydro-pyrrol-
-1-yl)-propionylamino]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-eth-
oxy}-ethoxy)-propionylamino]-propyl-oxy}-5-hydroxy-phenyl)-carbonyl]-amino-
}-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbo-
nyl}-amino)-propionic acid; and
(S)-3-[({3-[3-(3-{2-[2-(2-{2-[3-(2,5-Dioxo-2,5-dihydro-pyrrol-1-yl)-propi-
onylamino]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-propionylamino)-propyl-oxy]-5-h-
ydroxy-phenyl}-carbonyl)-amino]-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4-
,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid.
21. A pharmaceutical composition comprising a compound of claim 1
and a pharmaceutically acceptable carrier.
Description
PRIORITY TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 61/678,673, filed Aug. 2, 2012, and U.S.
Provisional Patent Application No. 61/591,297, filed Jan. 27, 2012,
the disclosures of which are incorporated herein by reference in
their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to the synthesis and reaction
of potent and selective small molecule integrin antagonists
containing appropriate linkers and functional groups for chemical
reaction with other molecules which contain reactive nucleophiles
such as thiols such that a covalent linkage is formed between a
moiety to be conjugated and the targeting entity. The small
molecule targeting antagonists bind to cognate receptor systems as
LFA-1 antagonists and/or dual LFA-1/MAC-1 antagonists to the ICAM-1
receptor. The covalently linked moiety includes small molecule
therapeutics, polymers, peptides, and oligonucleotides. Included
are 5'-thio-containing oligonucleotides for formation of
5'-thio-siRNA derivatives as a means to enable targeted delivery of
said siRNAs. Such derivatized siRNAs in conjunction with
appropriate transfection agents aid in the selective delivery of
siRNAs to cells which express such integrin receptors, thereby
preventing the expression of target genes through RNA interference
(RNAi).
BACKGROUND OF THE INVENTION
[0003] The lymphocyte function-associated antigen 1, also known as
LFA-1 is an integrin which is found on all T-cells and also on
B-cells, macrophages and neutrophils and is involved in recruitment
to the site of infection. It binds to ICAM-1 on antigen-presenting
cells and functions as an adhesion molecule. ICAM-1 (Inter-Cellular
Adhesion Molecule 1) also known as CD54 (Cluster of Differentiation
54) is a cell surface glycoprotein. Aberrant levels of LFA-1/ICAM-1
interactions are thought to be operative in inflammatory diseases
and disorders and therefore, the antagonism of such systems is
thought to be a means of therapy. Therefore, the targeting of high
affinity small molecules to these systems may provide a means to
selectively deliver therapeutics such as siRNA to cellular systems
that express the ICAM-1 receptor.
[0004] RNA interference is a well-known process in which the
translation of messenger RNA (mRNA) into protein is interfered with
by the association or binding of complementary or partially
complementary oligonucleotides such as small interfering RNA
(siRNA), short hairpin RNA(shRNA), micro RNA (miRNA), or antisense
oligonucleotides. siRNAs are double-stranded RNA molecules, usually
ranging from 19-25 nucleotides in length that associate with a set
of proteins in the cytoplasm known as RISC(RNA-induced silencing
complex). RISC ultimately separates the double stranded siRNA
allowing one strand to bind or associate with a complementary or
partially complementary portion of an mRNA molecule after which the
mRNA is destroyed by RISC or otherwise prevented from being
translated--consequently suppressing the expression of the encoded
protein or gene product.
[0005] One of the problems in using nucleic acids such as siRNA in
therapeutic applications (especially for systemic administration in
humans) has been in delivering the nucleic acids to: (1) particular
target tissues or cell types and (2) to the cytoplasm of those
cells (i.e., where the mRNA is present and translated into
protein). Part of the delivery problem is based on the fact that
nucleic acids are negatively charged and easily degraded
(especially if unmodified), efficiently filtered by the kidney, and
cannot be easily transported to the cytoplasm of the cells by
themselves. Thus, a significant amount of research has focused on
solving the delivery problem with various carriers and formulations
including liposomes, micelles, peptides, polymers, conjugates and
aptamers. See Ling et al, Advances in Systemic siRNA Delivery,
Drugs Future 34(9): 721 (September 2009). Some of the more
promising delivery vehicles have involved the use of lipidic
systems including lipid nanoparticles. See Wu et al., Lipidic
Systems for In Vivo siRNA Delivery, AAPS J. 11(4): 639-652
(December 2009); International Patent Application Publication No.
WO 2010/042877 by Hope et al ("Improved Amino Lipids And Methods
For the Delivery of Nucleic Acids"). However, a need remains for
further improved targeting of siRNA, as well as other substances
such as small molecules, peptides, other nucleic acids, fluorescent
moieties, and polymers to particular target cells and to the
cytoplasm of such cells.
SUMMARY OF THE INVENTION
[0006] The invention relates to compounds of formula I:
##STR00002##
wherein R1, R2, and n are defined in the detailed description and
claims. In particular, the present invention relates to the
compounds of formula I for the improved delivery of conjugated
moieties such as small molecules, peptides, nucleic acids,
fluorescent moieties, and polymers to target cells expressing the
integrin .alpha.4.beta.1 (Very Late Antigen-4) dimer, the
.alpha.V.beta.3 dimer, or the lymphocyte function-associated
antigen 1 (LFA-1) for various therapeutic and other applications.
The present invention also relates to methods of manufacturing and
using such compounds.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] Table 1 shows the composition of particular 5'-derivatized
siRNA single and double strands.
[0008] Table 2 shows analytical data for small molecule siRNA
conjugates.
[0009] Table 3 shows the siRNA sequences wherein the 5'-antisense
strand has been derivatized with Nu547.
[0010] Table 4 shows small molecule-siRNA conjugate potencies in
integrin antagonists assays and siRNA KD data.
[0011] Table 5 shows the identity, characterization and binding
potencies of FITC isomer labeled reagents.
[0012] FIG. 1 shows a histograph (Red Duplex-27 500 nM and Example
140 10 .mu.M; green Duplex-27).
[0013] FIG. 2 shows representative siRNA uptake image (Duplex-27
(500 nM).
[0014] FIG. 3 shows images of Jurkat cells with FITC conjugated
with Example FITC-5 (LFA-1 antagonist-labeled FITC) at 10
.mu.M.
[0015] FIG. 4 shows images of Jurkat cells with FITC conjugated
with Example FITC-14 (VLA-4 antagonist-labeled FITC) at 10 .mu.M.
The histograph indicates a shift in presence of the siRNA duplex
with a VLA-4 targeting element. In the presence of VLA-4 antagonist
example 140, this shift is oblated.
[0016] FIG. 5 shows the reduction of AHA1 expression in H1299 cells
when treated with siRNA duplexes which have been derivatized on the
5'-sense strand with an integrin targeting small molecule. The
y-axis indicates the observed expression level of AHA1. The lower
bar indicates a greater degree of knock-down (a higher degree of
siRNA transfection); a high bar, a lesser degree of knock-down
(i.e., a lesser degree of siRNA transfection). Duplexes in blue
have targeting modifications on the 5'-end of the sense strand;
those in pink have targeting modifications on the 5'-end of the
sense strand as well as Nu547 fluorophore attached to the 5'-end of
the antisense strand.
[0017] FIG. 6 shows the levels of GAPDH mRNA expression, a marker
of cell health. The similarity of the expression levels for those
cells treated with derivatized siRNA to that of the mock and
untreated cells is an indication of the lack of cellular toxicity
at the treatment concentration and duration.
DETAILED DESCRIPTION OF THE INVENTION
[0018] Unless otherwise indicated, the following specific terms and
phrases used in the description and claims are defined as
follows:
[0019] The term "moiety" refers to an atom or group of chemically
bonded atoms that is attached to another atom or molecule by one or
more chemical bonds thereby forming part of a molecule. For
example, the variables R1 and R2 of formula I refer to moieties
that are attached to the structure shown in formula I by a covalent
bond where indicated.
[0020] The term "conjugated moiety" refers to moiety which is a
therapeutic or useful compound, peptide, polymer, small molecule,
fluorescent moiety, oligonucleotide or nucleic acid. Examples
include drugs, therapeutic peptides, antisense oligonucleotides,
siRNA, and fluorescein isothiocyanate (FITC).
[0021] Unless otherwise indicated, the term "hydrogen" or "hydro"
refers to the moiety of a hydrogen atom (--H) and not H.sub.2.
[0022] The term "halogen" refers to a moiety of fluoro, chloro,
bromo or iodo.
[0023] The term "alkyl" refers to an aliphatic straight-chain or
branched-chain saturated hydrocarbon moiety having 1 to 25 carbon
atoms.
[0024] The term "TFA" refers to trifluoroacetic acid.
[0025] Unless otherwise indicated, the term "a compound of the
formula" or "a compound of formula" or "compounds of the formula"
or "compounds of formula" means any compound selected from the
genus of compounds as defined by the formula (Including any
pharmaceutically acceptable salt or ester of any such compound If
not otherwise noted).
[0026] The term "pharmaceutically acceptable salts" refers to those
salts which retain the biological effectiveness and properties of
the free bases or free acids, which are not biologically or
otherwise undesirable. Salts may be formed with inorganic acids
such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric
acid, phosphoric acid and the like, preferably hydrochloric acid,
and organic acids such as acetic acid, propionic acid, glycolic
acid, pyruvic acid, oxalic acid, maleic acid, malonic acid,
salicylic acid, succinic acid, fumaric acid, tartaric acid, citric
acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic
acid, ethanesulfonic acid, p-toluenesulfonic acid, N-acetylcystein
and the like. In addition, salts may be prepared by the addition of
an inorganic base or an organic base to the free acid. Salts
derived from an inorganic base include, but are not limited to, the
sodium, potassium, lithium, ammonium, calcium, and magnesium salts
and the like. Salts derived from organic bases include, but are not
limited to salts of primary, secondary, and tertiary amines,
substituted amines including naturally occurring substituted
amines, cyclic amines and basic ion exchange resins, such as
isopropylamine, trimethylamine, diethylamine, triethylamine,
tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine,
piperidine, polyamine resins and the like. Depending on the
substitution patterns, the compounds of the present invention may
also exist as zwitterions.
[0027] The compounds of the present invention can be present in the
form of pharmaceutically acceptable salts. The compounds of the
present invention can also be present in the form of
pharmaceutically acceptable esters (i.e., the methyl and ethyl
esters of the acids of formula I to be used as prodrugs). The
compounds of the present invention can also be solvated, i.e.
hydrated. The solvation can be affected in the course of the
manufacturing process or can take place i.e. as a consequence of
hygroscopic properties of an initially anhydrous compound of
formula I (hydration).
[0028] Compounds that have the same molecular formula but differ in
the nature or sequence of bonding of their atoms or the arrangement
of their atoms in space are termed "isomers." Isomers that differ
in the arrangement of their atoms in space are termed
"stereoisomers." Diastereomers are stereoisomers with opposite
configuration at one or more chiral centers which are not
enantiomers. Stereoisomers bearing one or more asymmetric centers
that are non-superimposable mirror images of each other are termed
"enantiomers." When a compound has an asymmetric center, for
example, if a carbon atom is bonded to four different groups, a
pair of enantiomers is possible. An enantiomer can be characterized
by the absolute configuration of its asymmetric center or centers
and is described by the R- and S-sequencing rules of Cahn, Ingold
and Prelog, or by the manner in which the molecule rotates the
plane of polarized light and designated as dextrorotatory or
levorotatory (i.e., as (+) or (-)-isomers respectively). A chiral
compound can exist as either individual enantiomer or as a mixture
thereof. A mixture containing equal proportions of the enantiomers
is called a "racemic mixture".
[0029] The term "a therapeutically effective amount" means an
amount of a compound that is effective to prevent, alleviate or
ameliorate symptoms of disease or prolong the survival of the
subject being treated. Determination of a therapeutically effective
amount is within the skill in the art. The therapeutically
effective amount or dosage of a compound according to this
invention can vary within wide limits and may be determined in a
manner known in the art. Such dosage will be adjusted to the
individual requirements in each particular case including the
specific compound(s) being administered, the route of
administration, the condition being treated, as well as the patient
being treated. The daily dosage can be administered as a single
dose or in divided doses, or for parenteral administration, it may
be given as continuous infusion.
[0030] The term "pharmaceutically acceptable carrier" is intended
to include any and all material compatible with pharmaceutical
administration including solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and other materials and compounds compatible with
pharmaceutical administration. Except insofar as any conventional
media or agent is incompatible with the active compound, use
thereof in the compositions of the invention is contemplated.
Supplementary active compounds can also be incorporated into the
compositions.
[0031] In detail, the present invention relates to the compounds of
formula I:
##STR00003##
or pharmaceutically acceptable salts or esters thereof; wherein n
is 1-24 and wherein:
[0032] R1 is selected from the group consisting of:
[0033] (1) a compound of the formula:
##STR00004##
[0034] (2) a compound of the formula:
##STR00005##
and
[0035] (3) a compound of the formula:
##STR00006## [0036] wherein Q is H or OH;
[0037] R2 is selected from the group consisting of:
[0038] (1) a compound of the formula:
##STR00007##
[0039] (2) a compound of the formula:
##STR00008##
[0040] (3) a compound of the formula:
##STR00009##
and
[0041] (4) a compound of the formula:
##STR00010## [0042] wherein R3 is a conjugated moiety and X
represents either sulfur or a compound of the formula:
##STR00011##
[0043] As used in the above structures, the symbol is used to
indicate where the structure or moiety is attached to the base
molecule by a covalent bond. In addition, the phrase "to PEG" or
"to S" or similar language used in combination with the above
symbol, indicates where or how the structure or moiety is attached
to the base molecule if there a multiple attachment points. For
example, if R2 is a compound of the formula:
##STR00012## [0044] wherein X is a compound of the formula:
##STR00013##
[0044] then the structure based upon formula I would be:
##STR00014## [0045] wherein R1, R3, and n are as defined in formula
I.
[0046] The present invention also relates to methods of
manufacturing and using the compounds of formula I as well as
pharmaceutical compositions containing such compounds. The
compounds of formula I are useful in improving the delivery of
small molecules, proteins, nucleic acids, polymers, fluorescent
markers, and other substances to target cells expressing ICAM-1
receptors. In particular embodiments, the present invention relates
to compositions and formulations containing the compounds of
formula I which are useful in delivering siRNA to the cytoplasm of
target cells expressing ICAM-1 receptors to inhibit the expression
of certain target proteins through RNA interference.
[0047] In more particular embodiments, the invention relates to the
use of the compounds of formula I for formulation to facilitate the
delivery of nucleic acids such as siRNA to tumor cells and other
cell types expressing ICAM-1 receptors. Furthermore, the use of the
compounds of formula I to synthesize delivery formulations to treat
inflammation and proliferative disorders, like cancers, is part of
the invention.
[0048] R1 represents small molecule integrin antagonists which
target the compounds of formula I to LFA-1 integrins, thereby
facilitating their delivery to cells that express such
receptors.
[0049] In particular embodiments, the small molecule integrin
antagonist targeting moieties of R1 are attached at a position such
that the affinity of binding of the small molecule to the integrin
is not substantially reduced relative to the free small molecule
integrin antagonist. The R1 moieties of formula I target the ICAM-1
receptor (via the LFA-1 or dual LFA-1/MAC-1 antagonists to the
ICAM-1 receptor).
[0050] In particular embodiments, R1 is an LFA-1 and/or dual
LFA-1/MAC-1 antagonists or ICAM-1 receptor targeting moiety of the
formula:
##STR00015##
[0051] or a pharmaceutically acceptable salt or ester thereof.
[0052] In other embodiments, R1 is an LFA-1 and/or dual LFA-1/MAC-1
antagonists or ICAM-1 receptor targeting moiety of the formula:
##STR00016##
[0053] or a pharmaceutically acceptable salt or ester thereof.
[0054] In other embodiments, R1 is an LFA-1 and/or dual LFA-1/MAC-1
antagonists or ICAM-1 receptor targeting moiety of the formula:
##STR00017##
[0055] or a pharmaceutically acceptable salt or ester thereof,
wherein Q is H or OH.
[0056] R2 may represent reactive moieties which can form covalent
linkages with therapeutic or other useful compounds or conjugated
moieties having strong nucleophiles such as thiol-containing
molecules. Examples of such reactive moieties include moieties
selected from the group consisting of:
##STR00018##
[0057] Alternatively, R2 may represent a moiety which is already
attached to a conjugated moiety such as a therapeutic or other
useful compound, protein, or oligonucleotide (R3). More
specifically, R2 may represent a moiety of the formula:
##STR00019## [0058] wherein R3 is a conjugated moiety and X
represents either sulfur or a compound of the formula:
##STR00020##
[0059] In particular embodiments, R3 represents an oligonucleotide.
In more specific embodiments, R3 represents the 5'-end of the sense
strand of an RNA molecule which may exist as a single strand or in
a duplex such as a siRNA molecule. Such siRNA molecules, also known
as RNAi agents, inhibit the expression of a target gene in a cell.
In specific embodiments, R3 is a siRNA molecule that consists
essentially of an oligoribonucleotide strand of between 15 and 30
nucleotides in length, wherein the 5' terminus of the sense
oligoribonucleotide strand is coupled to R2 as shown in the above
structures and is complementary to at least one portion of an mRNA
corresponding to the target gene. In other embodiments, R3 is an
oligonucleotide of DNA attached at its 5'-end. Such derivatized DNA
may exist as a single strand or as one strand hybridized with a
complementary strand of another oligonucleotide. The
oligonucleotide strands can be either unmodified or modified for
metabolic stability. Such modifications include, but are not
limited to, substitutions at specific positions on the phosphate
(e.g., phosphorothioate) and 2'-hydroxy (e.g., 2'-O-methyl and
2'-fluoro).
[0060] In particular embodiments, R2 of formula I represents
--X--S--CH.sub.2--R3 wherein R3 includes a sense strand of RNA as
shown below in formula 5 (based on formula I):
##STR00021##
[0061] wherein R1, n, and X are as defined in formula I.
[0062] In other particular embodiments, the sense strand may be
bound to an antisense strand.
[0063] In other specific embodiments, R2 represents
--X--S--CH.sub.2--R3 wherein R3 represents a small molecule or
protein, thereby forming a covalently linked, specifically targeted
entity of formula I.
[0064] In more specific embodiments, R2 represents
--X--S--CH.sub.2--R3 wherein R3 represents a therapeutic small
molecule or protein.
[0065] In other specific embodiments, R2 represents
--X--S--CH.sub.2--R3 wherein R3 represents a fluorescent moiety
useful for the visualization of these integrin receptor bindings
using cellular microscopy techniques.
[0066] In other specific embodiments, R2 represents
--X--S--CH.sub.2--R3 wherein R3 represents a polymer having
primary, reactive sulfides. More specifically, R3 may represent a
cationic polymer useful for the complexation and delivery of siRNA
to cell surfaces and the cytoplastic domains of cells.
[0067] In more particular embodiments, the present invention is
directed to compounds of formula I wherein R3 is one of the
structural isomers of fluorescein isothiocyanate (FITC) shown
below:
##STR00022##
[0068] In other more particular embodiments, the present invention
is directed to compounds of formula I wherein R3 is one of the
structural isomers of FITC-14 shown below:
##STR00023##
[0069] In other embodiments, the present invention is directed to a
compound of formula I wherein n is 9-13, preferably 12.
[0070] In more specific embodiments, the present invention is
directed to a compound of formula I selected from the group
consisting of one of the following compounds (or a pharmaceutically
acceptable salt or ester thereof):
LFA-1 Ligand Reagent 1
[0071]
(S)-3-{3-{2-[2-(2-{2-[3-(2,5-Dioxo-2,5-dihydro-pyrrol-1-yl)-propion-
ylamino]-ethoxy]-ethoxy}-ethoxy)-ethoxy]-propionylamino}-2-({2-[3-(3-hydro-
xy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}amino)propionic
acid;
LFA-1 Ligand Reagent 2
[0072]
(S)-3-{3-(2-{2-[2-(2-{2-[2-(2-{2-[3-(2,5-Dioxo-2,5-dihydro-pyrrol-1-
-yl)-propionylamino]-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethox-
y)-ethoxy]-propionylamino}-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-di-
methyl-pyrimidine-5-carbonyl}amino)propionic acid;
LFA-1 Ligand Reagent 3
[0073]
(S)-3-{3-[2-(2-{2-[2-(2-{2-[2-(2-{2-[2-(2-{2-[3-(2,5-Dioxo-2,5-dihy-
dro-pyrrol-1-yl)-propionylamino]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-e-
thoxy]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-propionylamino}-2-(-
{2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-p
yrimidine-5-carbonyl}amino)propionic acid;
LFA-1 Ligand Reagent 4
[0074]
(S)-3-{4-[4-(3-(2-{2-[2-(2-{2-[2-(2-{2-ethoxy)-ethoxy]-ethoxy}-etho-
xy)-ethoxy]-ethoxy}-ethoxy)-ethoxy]-amino-propoxy)-2,6-dichloro-benzoylami-
no]-phenyl}-2-[2-chloro-4-(3-hydroxy-benzylcarbamoyl)-benzoylamino]-propio-
nic acid-PEG 8;
LFA-1 Ligand Reagent 5
[0075]
S)-2-[2-Chloro-4-(3-hydroxy-benzylcarbamoyl)-benzoylamino]-3-(4-{2,-
6-dichloro-4-[3-(3-{2-[2-(2-{2-[3-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-prop-
ionylamino]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-propionylamino)-propoxy]-benzo-
ylamino}-phenyl)-propionic acid;
LFA-1 Ligand Reagent 6
[0076]
S)-2-[2-Chloro-4-(3-hydroxy-benzylcarbamoyl)-benzoylamino]-3-(4-{2,-
6-dichloro-4-[3-(3-{2-[2-(2-{2-2-[2-(2-{2-2-[2-(2-{2-[3-(2,5-dioxo-2,5-dih-
ydro-pyrrol-1-yl)-propionylamino]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy}--
ethoxy)-ethoxy]-ethoxy}-propionylamino)-propoxy]-benzoylamino}-phenyl)-pro-
pionic acid;
LFA-1 Ligand Reagent 7
[0077]
S)-2-[2-Chloro-4-(3-hydroxy-benzylcarbamoyl)-benzoylamino]-3-(4-{2,-
6-dichloro-4-[3-(3-{2-[2-(2-{2-2-[2-(2-{2-[3-(2,5-dioxo-2,5-dihydro-pyrrol-
-1-yl)-propionylamino]-ethoxy}-ethoxy)-ethoxy]-ethoxy]-ethoxy}-ethoxy)-eth-
oxy]-ethoxy}-propionylamino)-propoxy]-benzoylamino}-phenyl)-propionic
acid;
LFA-1 Ligand Reagent 8
[0078]
(S)-3-{[(3-{3-[3-(2-{2-[2-(2-{2-[2-(2-{2-[3-(2,5-Dioxo-2,5-dihydro--
pyrrol-1-yl)-propionylamino]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethox-
y]-ethoxy}-ethoxy)-propionylamino]-propyl-oxy}-phenyl)-carbonyl]-amino}-2--
({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-
-amino)-propionic acid;
LFA-1 Ligand Reagent 9
[0079]
(S)-3-{[(3-{3-[3-(2-{2-[2-(2-{2-[2-(2-{2-[3-(2,5-Dioxo-2,5-dihydro--
pyrrol-1-yl)-propionylamino]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-ethoxy)-ethox-
y]-ethoxy}-ethoxy)-propionylamino]-propyl-oxy}-5-hydroxy-phenyl)-carbonyl]-
-amino}-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-
-carbonyl}-amino)-propionic acid;
LFA-1 Ligand Reagent 10
[0080]
(S)-3-[({3-[3-(3-{2-[2-(2-{2-[3-(2,5-Dioxo-2,5-dihydro-pyrrol-1-yl)-
-propionylamino]-ethoxy}-ethoxy)-ethoxy]-ethoxy}-propionylamino)-propyl-ox-
y]-5-hydroxy-phenyl}-carbonyl)-amino]-2-({2-[3-(3-hydroxy-phenyl)-propylam-
ino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid.
[0081] In addition, the present invention relates to novel
compositions and formulations containing compounds of formula I for
the creation of nanoparticles upon combination with siRNA,
resulting in the improved delivery of nucleic acids such as siRNA
to the cytoplasm of target cells expressing LFA-1/ICAM-1 complexes.
In particular embodiments, the present invention is directed to a
siRNA formulation comprising: (1) a compound of formula I wherein
R2 includes a 5'-siRNA oligonucleotide; and (2) a polycationic
transfection agent.
[0082] The present invention also relates to methods of
manufacturing and using such compounds and compositions. The
compounds of formula I are useful as components of compositions or
formulations which improve the delivery of drugs, nucleic acids, or
other therapeutic compounds to tissues or cells expressing
LFA-1/ICAM-1 complexes. In particular embodiments, the present
invention relates to formulations containing the compounds of
formula I which are useful in delivering siRNA to the cytoplasm of
target cells LFA-1/ICAM-1 complexes to inhibit the expression of
certain proteins through RNA interference. In more particular
embodiments, the present invention relates to the compounds of
formula I and compositions containing such compounds that can
effectively deliver siRNA to tumor cells and other cell types
expressing ICAM-1 receptors for the treatment of cancer or
inflammatory diseases. Such compounds and compositions are more
efficacious and demonstrate improved knockdown capability compared
to similar formulations lacking the compounds of formula I.
General Synthesis of the Compounds of the Invention
[0083] Suitable processes for synthesizing compounds of formula I
are provided in the examples. Generally, compounds of formula I can
be prepared according to the schemes illustrated below. Unless
otherwise indicated, the variables n and R1 and R2 in the schemes
below are defined in the same manner as defined previously for the
genus of formula I.
General Synthesis of Maleimide-(PEG)n-Integrin Antagonists
Conjugating Agents
[0084] Compounds such as 26 in scheme 1 of various lengths of PEG
are commercially available (e.g., from Pierce BioScience). Such
compounds can also be made as by acylating the amino termini of PEG
amino acids with 3-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-propionic
acid under amide bond forming conditions, followed by formation of
reactive N-hydroxysuccinic esters by reaction of N-hydroxy succinic
acid under ester forming conditions. As shown in scheme 1, reacting
the compounds of 26 with compounds containing primary or secondary
amines such as 27 are conducted in aprotic or protic solvents in
the presence of basic amines such as DIEA (diisopropylethylamine)
at room temperature generating the PEGylated intermediates of
28.
##STR00024##
[0085] Compounds such as 29 in scheme 2 for which R4 is thioacetyl
or 2-dithiopyridyl and having PEG moieties of various lengths are
also commercially available (e.g., from Pierce BioScience).
Reaction of compounds having the structure of 29 with compounds
containing primary or secondary amines such as 27 are conducted in
aprotic or protic solvents in the presence of basic amines such as
DIEA (diisopropylethylamine) at room temperature generating the
PEGylated intermediates of 30.
##STR00025##
[0086] As a specific not limiting example for this invention,
intermediate 26 is reacted with 31 to produce the maleimide
intermediate of 32 as shown in Scheme 3:
##STR00026##
[0087] In a similar manner, intermediate 26 can be reacted with 33
to produce the maleimide intermediate of 34 as shown in Scheme
4:
##STR00027##
[0088] In a similar manner, intermediate 29 can be reacted with 35
to produce the intermediate of 36 as shown in Scheme 5 in which R4
represents either thioacetyl or 2-dithiopyridyl:
##STR00028##
[0089] In a similar manner, intermediate 29 can be reacted with 37
to produce intermediate of 38 as shown in Scheme 6 in which R4
represents either thioacetyl or 2-dithiopyridyl:
##STR00029##
[0090] For compounds of general structure 26 or 29, different PEG
lengths are available or easily made by those skilled in the art;
preferably n=8-24. This topic has been thoroughly reported and
reviewed (e.g., Chemistry for peptide and protein PEGylation,
Advanced Drug Delivery Reviews Volume 54, Issue 4, 17 Jun. 2002,
Pages 459-476).
[0091] Intermediate 31 can be synthesized in a manner similar to
that which has been reported (e.g., Sidduri, A. et al. Bioorganic
& Medicinal Chemistry Letters, 2002, 12, 2475-2478) as shown in
Scheme 7:
##STR00030## ##STR00031##
[0092] Specifically, as shown in Scheme 7, intermediate 41 was
created from commercially available
(S)-3-[4-nitrophenyl]-2-tert-butoxycarbonylamino-propionic acid 40.
The nitro group of commercially available starting material 40 in a
methanol solution was reduced with zinc dust in the presence of
ammonium chloride at room temperature over the course of several
hours, resulting in aniline 41. Other methods for nitro reduction
are known to those skilled in the art. Aniline 41 was acylated with
benzoyl halide derivatives such as 2,6-dichlorobenzoyl chloride 42
in aprotic solvent such as dichloromethane in the presence of a
base such as di-isopropyl-ethyl amine at room temperature. In this
manner, amide 43 was formed. The t-butylcarbonyl (Boc) amine
protecting group was removed according to standard methods known to
those skilled in the art, such as by treatment with an HCl solution
in dioxane at room temperature; this resulted in hydrochloride 44.
Hydrochloride 44 was treated with amide bond forming conditions
(also well known to those skilled in the art) in the presence of
known 1-(2-azido-ethyl)-cyclopentanecarboxylic acid 45 resulting in
the production of di-amide 46. The azide group of intermediate 46
was reduced by treatment with tri-alkyl phosphine in an aprotic
solvent such as tetrahydrofuran at room temperature. Further, the
methyl ester was saponified by treatment with sodium hydroxide in a
solvent mixture such as ethanol and tetrahydrofuran at an elevated
temperature such as 50.degree. C. and for 15 hours. This process
resulted in the formation of intermediate 31 which may also be
presented as a zwitterion.
[0093] Attachment of the PEG moiety is also possible with
intermediate 39, which is synthesized as shown in Scheme 8.
Specifically, 3,5-dichlorophenol 47 is protected with
tri-isopropylsilylchloride in the presence of a base such as
imidazole in a polar aprotic solvent such as DMF before reaction
with a strong base such as butyl lithium in anhydrous
tetrahydrofuran at low temperatures such as -78 degrees C. The
resulting lithium complex is quenched with carbon dioxide added in
the form of dry ice resulting in intermediate 48, a benzoic acid
derivative. Intermediate 48 is then chlorinated to form the acyl
chloride by treatment in an aprotic solvent such as toluene with
sulfonyl chloride (SOCl.sub.2). At this time, the acyl chloride is
then reacted with amine hydrochloride 49 in the presence of base
such as di-isopropylethyl amine (DIPEA) in aprotic solvent such as
dichloromethane (DCM), thereby forming intermediate 50. The silyl
protecting group of intermediate 50 is removed by treatment with
tetrabutyl ammonium fluoride (TBAF) in a protic solvent such as
tetrahydrofuran at room temperature. This phenol intermediate is
reacted in the presence of a base such as potassium carbonate
(K.sub.2CO.sub.3) in an aprotic solvent such as dimethylformamide
(DMF) with 3-N-t-butyl-carbomate-1-bromopropane. In this manner
intermediate 52 is formed which upon deprotection with
trifluoroacetic acid (TFA) and subsequent hydrolysis with a base
such as sodium hydroxide in protic solvent such as ethanol forms
intermediate 39:
##STR00032##
Synthesis of LFA-1 Antagonists Derivatizing Agents
[0094] Small molecules which target the LFA-1/ICAM interaction,
thereby providing a means of targeting cells which express the ICAM
system is shown below in Schemes 11, 12, 13, and 14. As shown in
Scheme 11, the primary amide of 3-(3-methoxy)-propanoic acid ester
70 is formed and reduced under standard conditions known to those
skilled in the art. Separately, dihydropyrimidine is formed using a
Bignelli reaction with urea, acetaldehyde, and 3-oxo-butanoic acid
ethyl ester. The pyrimidine of this product is formed by treatment
of dihydropyrimidine with 50% nitric acid, resulting in
4,6-dimethyl-2-hydroxy-pyrimidine-5-carboxylic acid ethyl ester.
The chloride of this substance is formed by reaction with
POCl.sub.3 (phosphorus oxychloride) forming 72. Amine 71 is reacted
with chloride 72, forming secondary amine ester 73. At this point,
the methoxy group is removed by treatment with a Lewis acid such as
boron tribromide in an aprotic solvent to form phenol 74. This
phenol 74 is saponified in the presence of an aqueous base followed
by application of amide coupling conditions in the presence of
S-3-N-t-butyl-carbamate-2-carboxy-diaminopropane hydro chloride
(H-DAP(Boc)OMe hydrochloride) thereby forming Intermediate 77. The
Boc protecting group is removed under standard conditions followed
by saponification of the methyl ester to form the ICAM-1 targeting
small molecule 21.
[0095] Reaction Scheme 11 for the Following Examples:
[0096] LFA-1 Ligand Reagent 1, LFA-1 Ligand Reagent 2, LFA-1 Ligand
Reagent 3:
##STR00033##
[0097] Other small molecules which target the LFA-1/ICAM
interaction, thereby providing a means of targeting cells which
express the ICAM system is shown below in Scheme 12. Specifically,
2,6-dichloro-4-triisopropylsilanyloxy-benzoic acid 79 in a aprotic
solvent such as toluene is treated with chlorinating reagent
thionyl chloride under reflux conditions. Upon work-up, the acyl
chloride is then treated with a base such as di-isopropylethylamine
and with
(S)-3-(4-amino-phenyl)-2-tert-butoxycarbonylamino-propionic acid
methyl ester 80 thereby creating amide 81. The Boc amino protecting
group is removed under standard conditions and the resulting
primary amine 83 is coupled under standard amide bond forming
reaction conditions. The methyl ester of 84 has been reported in WO
01/58853; silyl protection of methyl ester 84 was performed by
standard conditions well known to those skilled in the art. After
coupling and deprotection, amide 88 was then treated with
(3-bromo-propyl)-carbamic acid tert-butyl ester. The Boc protecting
group is removed under standard conditions followed by
saponification of the methyl ester to form the ICAM-1 targeting
small molecule 22.
[0098] Reaction Scheme 12 for the Following Examples:
[0099] LFA-1 Ligand Reagent 4, LFA-1 Ligand Reagent 5, LFA-1 Ligand
Reagent 6, LFA-1 Ligand Reagent 7
##STR00034## ##STR00035##
[0100] In a similar manner, the production of other small molecules
which target the LFA-1/ICAM interaction, thereby providing a means
of targeting cells which express the ICAM system is shown below in
Scheme 13. Specifically, 3-hydroxymethylbenzoate is alkylated with
(3-bromo-propyl)-carbamic acid tert-butyl ester under basic
conditions such as in the presence of potassium carbonate solvent
mixtures such as acetone and DMF thereby creating intermediate 91.
The methyl ester of 91 is saponified and the resulting free acid 92
is coupled under standard amide bond forming conditions with
intermediate 93 (Scheme 11) to provide intermediate 94. The Boc
protecting group is removed under standard conditions followed by
saponification of the methyl ester to form the ICAM-1 targeting
small molecule 96.
[0101] Reaction Scheme 13 for the Following Example:
[0102] LFA-1 Ligand Reagent 8
##STR00036##
[0103] A similar sequence of reactions is used to create compound
102 shown below in Scheme 14 which targets the LFA-1/ICAM
interaction, thereby providing a means of targeting cells which
express the ICAM system. Instead of starting with
3-hydroxymethylbenzoate, the starting material of
3,5-dihydroxymethylbenzoate 97 is used in a similar sequence to
create intermediate 103 also shown in Scheme 14.
[0104] Reaction Scheme 14 for the Following Examples:
[0105] LFA-1 Ligand Reagent 9, LFA-1 Ligand Reagent 10
##STR00037##
Utility
[0106] The compounds of formula I are useful in delivering
conjugated moieties such as therapeutics, small molecules,
peptides, nucleic acids, fluorescent moieties, and polymers to
target cells expressing LFA-1 integrin receptor complexes for
various therapeutic and other applications. Accordingly, the
compounds of formula I may be used for treating various diseases
and conditions that are associated with the expression or
overexpression of LFA-1. Such diseases and conditions may include
inflammation, cancer, and metabolic related diseases.
[0107] In particular embodiments, the present invention comprises a
method of treating or preventing cancer in a mammal (preferably a
human) in need of such treatment, wherein the method comprises
administering a therapeutically effective amount of a compound of
formula I. Such compositions can be administered in a fashion
consistent with good medical practice. Factors for consideration in
this context include the particular disorder being treated, the
particular mammal being treated, the clinical condition of the
individual patient, the cause of the disorder, the site of delivery
of the agent, the method of administration, the scheduling of
administration, and other factors known to medical practitioners.
The "effective amount" of the compound to be administered will be
governed by such considerations as the minimum amount necessary to
treat or prevent the disease or condition (e g inhibit the
expression of a target protein) and avoid unacceptable toxicity.
For example, such amount may be below the amount that is toxic to
normal cells, or the mammal as a whole. The compositions containing
a compound of formula I of the invention may be administered by
parenteral, intraperitoneal, and intrapulmonary administration.
Parenteral infusions include intramuscular, intravenous,
intraarterial, intraperitoneal, or subcutaneous administration.
EXAMPLES
[0108] The invention will be more fully understood by reference to
the following examples. They should not, however, be construed as
limiting the scope of the invention.
[0109] Reagents were purchased from Aldrich, Sigma, and Pierce
BioScience or other suppliers as indicated below and used without
further purification. The purification of multi-milligram to
multi-gram scale was conducted by methods known to those skilled in
the art such as elution of silica gel flash column. Preparative
flash column purifications were also effected in some cases by use
of disposable pre-packed multigram silica gel columns (RediSep)
eluted with a CombiFlash system. Biotage.TM. and ISCO.TM. are also
flash column instruments that may be used in this invention for
purification of intermediates.
[0110] For the purpose of judging compound identity and purity,
LC/MS (liquid chromatography/mass spectroscopy) spectra were
recorded using the following system. For measurement of mass
spectra, the system consists of a Micromass Platform II
spectrometer: ES Ionization in positive mode (mass range: 150-1200
amu). The simultaneous chromatographic separation was achieved with
the following HPLC system: ES Industries Chromegabond WR C-18 3 u
120 .ANG. (3.2.times.30 mm) column cartridge; Mobile Phase A: Water
(0.02% TFA) and Phase B: Acetonitrile (0.02% TFA); gradient 10% B
to 90% B in 3 minutes; equilibration time of 1 minute; flow rate of
2 mL/minute. In some cases, ammonium acetate at 20 millimolar
concentration was used as a modifier for effective ionization
during preparative HPLC. In such cases, the ammonium salt was
isolated.
[0111] For some separations, the use of super critical fluid
chromatography may also be useful. Super critical fluid
chromatography separations were performed using a Mettler-Toledo
Minigram system with the following typical conditions: 100 bar,
30.degree. C., 2.0 mL/min eluting a 12 mm AD column with 40% MeOH
in super critical fluid CO.sub.2. In the case of analytes with
basic amino groups, 0.2% isopropyl amine was added to the methanol
modifier.
[0112] Compounds were characterized either by .sup.1H-NMR using a
Varian Inova 400 MHz NMR Spectrometer or a Varian Mercury 300 MHz
NMR Spectrometer as well as by high resolution mass spectrometry
using a Bruker Apex-II high-resolution 4.7 T FT-Mass Spectrometer.
Final compounds were also characterized by high resolution mass
spectrometry using a LTQ CL Orbitrap sold by Thermo Electron.
[0113] Abbreviations used herein are as follows:
AIBN 2,2'-azobisisobutyronitrile Bu butyl DCE 1,2-dichloroethane
DCM dichloromethane DBU 1,8-diazabicyclo[5.4.0]undec-7-ene DIAD
diisopropyl azodicarboxylate DIEA diethylamine DIPEA
diisopropylethylamine
DMF N,N-dimethylformamide
[0114] DMSO dimethylsulfoxide EDC-HCl
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride EtOAc
ethyl acetate EtOH ethyl alcohol FCC flash column chromatography h
hour HBTU
O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluroniumhexafluorophosphate
HOBt hydroxybenzotriazole HPLC high pressure liquid chromatography
HRMS high resolution mass spectra LRMS low resolution mass spectra
LC liquid chromatography
L-Pro L-proline
[0115] MCPBA meta-chloroperoxybenzoic acid MeOH methyl alcohol MW
microwave
NIS N-iodosuccinimide
NBS N-bromosuccinimide
[0116] NMP 1-methyl-2-pyrrolidinone PdCl.sub.2(dppf)
[1,1'-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) PEGn
Polyethylene glycol repeating n times (e.g.,
PEG2=--OCH2CH2OCH2CH2-) PG protecting group PyBroP
bromo-tris-pyrrolidino-phosphonium hexafluorophosphate rt room
temperature TBAF tetrabutylammonium fluoride TBDMS
tert-butyl-dimethylsilyl TBTU
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate TMS trimethylsilyl TEA triethylamine TFA
trifluoroacetic acid THF tetrahydrofuran TLC thin layer
chromatography TPP triphenylphosphine
Synthesis of Small Molecule LFA-1 Antagonists and/or Dual
LFA-1/MAC-1 Antagonists to the ICAM-1 Receptor for Use as Targeting
Agents
Part 1
Preferred Intermediates
Preparation of 3-(3-methoxy-phenyl)-propionamide
##STR00038##
[0118] A solution of 3-(3-methoxy-phenyl)-propionic acid (15 g,
83.2 mmol) and 4-methyl-morpholine (10.1 ml, 91.56 mmol) in THF
(150 ml) was cooled to 0.degree. C. (ice-water bath), and
iso-propyl chloroformate (1M in toluene, 91.6 ml, 91.56 mmol) was
added over 20 minutes. The mixture was stirred for another 30
minutes at 0.degree. C., followed by dropwise addition of 7N
NH.sub.3/MeOH (24 ml, 168 mmol). The mixture was allowed to warm up
to room temperature and stirred for 2 h. It was quenched with 10%
aq K.sub.2CO.sub.3 and extracted with EtOAc. The organic extracts
were combined, washed with water and brine, dried over sodium
sulfate, filtered and evaporated to give the desired amide (11.15
g, 75% yield). MS m/e 179.9 (M+H.sup.+).
Preparation of 3-(3-methoxy-phenyl)-propylamine
##STR00039##
[0120] BH.sub.3 in THF (2.2 g, 188 mmol) was added at room
temperature to a solution of 3-(3-methoxy-phenyl)-propionamide
(11.15 g, 62.26 mmol) in THF (100 ml). The solution was heated to
reflux for 4 h, cooled to room temperature and quenched with MeOH
(50 ml). The solution was heated to reflux for 30 min,
concentrated, treated with water, and extracted with EtOAc. The
extract was washed with 10% aqK.sub.2CO.sub.3, water and brine,
dried over Na.sub.2SO.sub.4, filtered and evaporated to give title
compound (9.26 g, 90% yield). MS m/e 165.9 (M+H.sup.+).
Preparation of
4,6-Dimethyl-2-hydroxy-1,6-dihydro-pyrimidine-5-carboxylic acid
ethyl ester
##STR00040##
[0122] A mixture of 3-oxo-butanoic acid ethyl ester (16.27 g, 125
mmol), acetaldehyde (5.51 g, 125 mmol), urea (7.51 g, 125 mmol),
and glacial acetic acid (20 drops) in ethanol (35 ml) was heated to
90.degree. C. overnight in a 350 ml pressure flask. The mixture was
diluted with water. The precipitate was collected by filtration,
washed with water and air-dried to afford the desired product
(17.68 g, 71% yield). MS m/e 198.8 (M+H.sup.+).
Preparation of 4,6-dimethyl-2-hydroxy-pyrimidine-5-carboxylic acid
ethyl ester
##STR00041##
[0124] 4,6-Dimethyl-2-hydroxy-1,6-dihydro-pyrimidine-5-carboxylic
acid ethyl ester (34.63 g, 174.7 mmol) was added in portions to an
ice-cooled solution of 50% nitric acid (120 ml) over 5 minutes. The
solution was stirred at 0.degree. C. for 10 minutes, poured into
ice water (500 ml), neutralized with solid K.sub.2CO.sub.3 and
extracted with chloroform. The combined organic layers were washed
with water and brine, dried over Na.sub.2SO.sub.4, filtered, and
concentrated to afford title compound (21.9 g, 71% yield). MS m/e
197.1 (M+H.sup.+).
Preparation of 2-Chloro-4,6-dimethyl-pyrimidine-5-carboxylic acid
ethyl ester
##STR00042##
[0126] To a solution of POCl.sub.3 (106 ml) and DIEA (65 ml) was
added 4,6-dimethyl-2-hydroxy-pyrimidine-5-carboxylic acid ethyl
ester (21.9 mg, 111.6 mmol). The mixture was heated to 110.degree.
C. for 2 h. Excess POCl.sub.3 and DIEA were removed by evaporation
under reduced pressure. The residue was dissolved in EtOAc (1.21)
and treated with decolorizing carbon. After filtration, the
solution was washed with 1N NaOH, water and brine. The organic
layer was dried over Na.sub.2SO.sub.4. filtered and concentrated.
The crude residue was purified by flash chromatography with a 0-30%
EtOAc in hexane gradient to afford the desired product (9.33 g, 39%
yield).
Preparation of
2-[3-(3-methoxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carboxylic
acid ethyl ester
##STR00043##
[0128] A mixture of 3-(3-methoxy-phenyl)-propylamine (2.31 g, 13.98
mmol), 2-chloro-4,6-dimethyl-pyrimidine-5-carboxylic acid ethyl
ester (2 g, 9.32 mmol) in EtOH (12 ml) was microwaved at
160.degree. C. for 1.5 h. The reaction mixture was cooled to room
temperature, quenched with 10% K.sub.2CO.sub.3 and extracted with
EtOAc. The organic layer was washed with brine, dried over
Na.sub.2SO.sub.4, filtered and concentrated. The residue was
purified by flash chromatography with 30% EtOAC in hexane to afford
the desired product (2.42 g, 76% yield). MS m/e 344.1
(M+H.sup.+).
Preparation of
2-[3-(3-hydroxy-phenyl)-propylamino)-4,-dimethyl-pyrimidine-5-carboxylic
acid ethyl ester
##STR00044##
[0130] A solution of
2-[3-(3-methoxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carboxylic
acid ethyl ester (2.42 g, 7.05 mmol) DCM (50 ml) was cooled in an
ice-water bath and BBr.sub.3/DCM (1M, 14.1 ml, 14.1 mmol) was added
dropwise. The resulting solution was allowed to warm up to room
temperature and stirred at room temperature for 2 h. The solution
was quenched with ice water and extracted with DCM. The organic
layers were combined, washed with water and brine, dried over
MgSO.sub.4, filtered, and concentrated to afford the desired
product (2 g, 86% yield). MS m/e 330.1 (M+H.sup.+).
Preparation of
(S)-3-tert-butoxycarbonylamino-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4-
,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl
ester
##STR00045##
[0132] A solution of
2-[3-(3-hydroxy-phenyl)-propylamino]-4,5-dimethyl-pyrimidine-5-carboxylic
acid ethyl ester (2.0 g, 6.0 mmol) in dioxane (30 ml) was treated
with a solution of lithium hydroxide monohydrate (6.3 g, 150 mmol)
in water (30 ml). The mixture was stirred at 90.degree. C. for 12
h, then cooled to room temperature and quenched with aqueous
potassium hydrogen sulfate to adjust the pH to .about.2-4. The
resulting solution was extracted with EtOAc. The organic extracts
were combined, washed with brine, dried over sodium sulfate,
filtered and concentrated to give the acid (1.76 g) which was not
purified but directly submitted to the next step. To a solution of
2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carboxy-
lic acid (1.76 g, 5.84 mmol) in anhydrous DMF (60 ml) was added
Et.sub.3N (2.5 ml, 7.0 mmol), HBTU (2.66 g, 7.01 mmol), HOBT (0.95
g, 7.01 mmol), and H-DAP(Boc)OMe hydrochloride (1.79 g, 7.01 mmol).
The mixture was stirred at room temperature for 3 h, diluted with
brine (200 ml) and extracted with ethyl acetate. The combined
organic layers were washed with 1:1 saturated sodium
bicarbonate/brine and brine, then dried over Na.sub.2SO.sub.4,
filtered, and concentrated under reduced pressure. The crude
residue was purified by flash chromatography with a 40-100% EtOAc
in hexane gradient to give the title compound (2.66 g, 91% yield).
MS m/e 501.9 (M+H.sup.+).
Preparation of
(S)-3-Amino-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimid-
ine-5-carbonyl}-amino)-propionic acid methyl ester
hydrochloride
##STR00046##
[0134] To a solution of
(S)-3-tert-Butoxycarbonylamino-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4-
,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl
ester (2.66 g, 5.30 mmol) in MeOH (10 ml) was added 4.0 M HCl in
dioxane (20 mL). After one hour the mixture was concentrated and
azeotroped with MeOH. The product was triturated with ether,
filtered, and washed with ether to afford the title compound (2.16
g, 93% yield). MS m/e 401.9 (M+H.sup.+).
Preparation of
(S)-3-amino-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimid-
ine-5-carbonyl}-amino)-propionic acid hydrochloride; LFA-1 Ligand
1
##STR00047##
[0136]
S)-3-Amino-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-py-
rimidine-5-carbonyl}-amino)-propionic acid methyl ester
hydrochloride (50 mg, 0.114 mmol) was added to aqueous solution of
LiOH (13 mg, 0.57 mmol in 2 mL of water) and the resulting
suspension was stirred at room temperature overnight. Then the
reaction mixture was neutralized with 1N hydrochloric acid and
lyophilized. This material was used with any additional
purification for the next step.
Preparation of 3-(3-tert-butoxycarbonylamino-propoxy)-benzoic acid
methyl ester
##STR00048##
[0138] 3-Hydroxymethylbenzoate (500 mg, 3.29 mmol),
(3-bromo-propyl)-carbamic acid tert-butyl ester (861 mg, 1.1 eq.)
and potassium carbonate (2.3 g, 5 eq.) were combined in a mixture
of acetone (10 mL) and DMF (10 mL). The reaction mixture was
stirred at 75.degree. C. overnight. The insoluble material was
filtered and discarded and the filtrate was concentrated under
reduced pressure, diluted with ethyl acetate and washed with water
and brine, followed by drying over anhydrous sodium sulfate. Flash
chromatography on silica gel using ethyl acetate and hexanes
afforded 900 mg of the title compound. HRMS m/e 332.1466
(M+Na).sup.+
Preparation of 3-(3-tert-butoxycarbonylamino-propoxy)-benzoic
acid
##STR00049##
[0140] To a solution of
3-(3-tert-butoxycarbonylamino-propoxy)-benzoic acid methyl ester
(900 mg) in methanol (3 mL) was added a solution of LiOH (334 mg, 5
eq.) in water (3 mL) and the resulting reaction mixture was stirred
at 45.degree. C. overnight. Then the reaction mixture was acidified
with 1 N HCl to pH 3 and immediately extracted with ethyl acetate.
The organic phase was washed with brine and dried over anhydrous
sodium sulfate. It was then concentrated under reduced pressure and
crystallized from ethyl acetate to afford 600 mg of the title
compound.
[0141] HRMS m/e 318.1311 (M+Na).sup.+
Preparation of
(S)-3-[3-(3-tert-butoxycarbonylamino-propoxy)-benzoylamino]-2-({2-[3-(3-h-
ydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-pro-
pionic acid methyl ester
##STR00050##
[0143] To a solution of
3-(3-tert-butoxycarbonylamino-propoxy)-benzoic acid (57 mg, 0.193
mmol) in DMF (1 mL) were added HBTU (78 mg, 1.05 eq.), DIEA (172
.mu.L, 5 eq.) and
(S)-3-amino-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimid-
ine-5-carbonyl}-amino)-propionic acid methyl ester hydrochloride
(100 mg, 0.194 mmol). The resulting reaction mixture was stirred at
room temperature for 4 h. It was then diluted with ethyl acetate,
washed with water and brine and dried over anhydrous sodium
sulfate. Flash chromatography on silica gel using
methanol/methylene chloride afforded 97 mg of the title
compound.
[0144] HRMS m/e 679.3447 (M+H).sup.+
Preparation of
(S)-3-[3-(3-amino-propoxy)-benzoylamino]-2-({2-[3-(3-hydroxy-phenyl)-prop-
ylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid
methyl ester hydrochloride
##STR00051##
[0146] Trimethylsilyl chloride (177 .mu.L) was added to methanol (2
mL) and the resulting mixture was stirred at room temperature for 5
min. Then it was added to
(S)-3-[3-(3-tert-butoxycarbonylamino-propoxy)-benzoylamino]-2-({2-[3-(3-h-
ydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-pro-
pionic acid methyl ester (94.6 mg, 0.139 mmol) and the resulting
reaction mixture was stirred at room temperature over the weekend.
Then it was concentrated under reduced pressure and triturated with
diethyl ether to afford 84.8 mg of the title compound. HRMS m/e
579.2925 (M+H).sup.+
Preparation of
(S)-3-[3-(3-amino-propoxy)-benzoylamino]-2-({2-[3-(3-hydroxy-phenyl)-prop-
ylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid
LAF-1 Ligand 2
##STR00052##
[0148]
(S)-3-[3-(3-Amino-propoxy)-benzoylamino]-2-({2-[3-(3-hydroxy-phenyl-
)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic
acid methyl ester hydrochloride (82.6 mg, 0.134 mmol) was dissolved
in methanol (1 mL) and 2 M NaOH (336 .mu.L, 5 eq.) and the
resulting reaction mixture was stirred at room temperature
overnight. Then, it was neutralized with 1 N HCl, lyophilized and
used for the next step without further purification. MS m/e 565.5
(M+H).sup.+
Preparation of
3-(3-tert-butoxycarbonylamino-propoxy)-5-hydroxy-benzoic acid
methyl ester
##STR00053##
[0150] 3,5-Dihydroxymethylbenzoate (1.8 g, 10.7 mmol),
(3-bromo-propyl)-carbamic acid tert-butyl ester (1.3 g, 5.46 mmol)
and potassium carbonate (1.5 g, 10.8 mmol) were combined in a
mixture of acetone (50 mL) and DMF (50 mL). The reaction mixture
was stirred at 75.degree. C. overnight. The crude reaction mixture
was concentrated under reduced pressure, diluted with ethyl acetate
and washed with water and brine, followed by drying over anhydrous
sodium sulfate. Flash chromatography on silica gel using ethyl
acetate and hexanes afforded 462 mg of the title compound. HRMS m/e
348.1417 (M+Na).sup.+
Preparation of
3-(3-tert-butoxycarbonylamino-propoxy)-5-hydroxy-benzoic acid
##STR00054##
[0152] To a solution of
3-(3-tert-butoxycarbonylamino-propoxy)-5-hydroxy-benzoic acid
methyl ester (1.2 g 3.69 mmol) in 2M NaOH (9.2 mL, 5 eq.) was added
water (20 mL) and the resulting reaction mixture was stirred at
room temperature overnight. Then the reaction mixture was
neutralized with 1 N HCl and immediately extracted with ethyl
acetate. The organic phase was washed with brine and dried over
anhydrous sodium sulfate. It was then concentrated under reduced
pressure to afford 1.0 g of the title compound. MS m/e 211.8
(M+H-Boc).sup.+
Preparation of
3-(3-tert-butoxycarbonylamino-propoxy)-5-hydroxy-benzoic acid
2,5-dioxo-pyrrolidin-1-yl ester
##STR00055##
[0154] To a cooled solution of
3-(3-tert-butoxycarbonylamino-propoxy)-5-hydroxy-benzoic acid (500
mg, 1.606 mmol) and N-hydroxysuccinimide (185 mg, 1 eq.) in THF (20
mL) was added DCC (332 mg, 1 eq.). The cooling bath was removed
after 1 h. The insoluble material was filtered and discarded. The
filtrate was concentrated under reduced pressure and the crude
material was purified by flash chromatography on silica gel using
ethyl acetate and hexanes to afford 602 mg of the title compound.
HRMS m/e 431.1426 (M+Na).sup.+
Preparation of
(S)-3-[3-(3-tert-butoxycarbonylamino-propoxy)-5-hydroxy-benzoylamino]-2-(-
{2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}--
amino)-propionic acid methyl ester
##STR00056##
[0156] To a solution of
(S)-3-amino-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimid-
ine-5-carbonyl}-amino)-propionic acid methyl ester hydrochloride
(476.4 mg, 0.924 mmol) in DMF (5 mL) were added DIEA (321 .mu.L, 3
eq.) and 3-(3-tert-butoxycarbonylamino-propoxy)-5-hydroxy-benzoic
acid 2,5-dioxo-pyrrolidin-1-yl ester (377 mg, 1 eq.). The resulting
reaction mixture was stirred at room temperature for 2 h. Then it
was diluted with ethyl acetate and washed with water and brine and
dried over anhydrous sodium sulfate. The crude material was
purified by flash chromatography on silica gel using
methanol/methylene chloride to afford 301 mg of the title
compound.
[0157] HRMS m/e 695.3395 (M+H).sup.+
Preparation of
(S)-3-[3-(3-amino-propoxy)-5-hydroxy-benzoylamino]-2-({2-[3-(3-hydroxy-ph-
enyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic
acid methyl ester hydrochloride
##STR00057##
[0159] Trimethylsilyl chloride (548 .mu.L) was added to methanol (5
mL) and the resulting solution was stirred at room temperature for
1 min. Then
(S)-3-[3-(3-tert-butoxycarbonylamino-propoxy)-5-hydroxy-benzoylamino-
]-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbo-
nyl}-amino)-propionic acid methyl ester (299.6 mg, 0.431 mmol) was
added and stirring at room temperature was continued overnight.
Methanol was removed under reduced pressure and the residue was
triturated with diethyl ether to afford 272 mg of the title
compound. HRMS m/e 595.2875 (M+H).sup.+
Preparation of
(S)-3-[3-(3-amino-propoxy)-5-hydroxy-benzoylamino]-2-({2-[3-(3-hydroxy-ph-
enyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic
acid; LFA-1 Ligand 3
##STR00058##
[0161]
(S)-3-[3-(3-amino-propoxy)-5-hydroxy-benzoylamino]-2-({2-[3-(3-hydr-
oxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propio-
nic acid methyl ester hydrochloride (100 mg, 0.158 mmol) was
dissolved in a mixture of water (1 mL) and methanol (1 mL) and then
2 N NaOH was added (400 .mu.L, 5 eq.). The reaction mixture was
stirred at room temperature for 3 h. Then it was neutralized with 1
N HCl, lyophilized and used for the next step without further
purification. MS m/e 581.1 (M+H).sup.+
Preparation of
(S)-2-tert-butoxycarbonylamino-3-[4-(2,6-dichloro-4-triisopropylsilanylox-
y-benzoylamino)-phenyl]-propionic acid methyl ester
##STR00059##
[0163] To a solution of
2,6-dichloro-4-triisopropylsilanyloxy-benzoic acid (50 mg, 0.138
mmol) in toluene (2 mL) was added thionyl chloride (50 .mu.L, 0.69
mmol). The resulting solution was refluxed for 2 h. Then thionyl
chloride and toluene were removed under reduced pressure. The oily
residue was redissolved in methylene chloride (3 mL) and cooled to
0.degree. C. Then DIEA (72 .mu.L, 0.414 mmol) and
(S)-3-(4-amino-phenyl)-2-tert-butoxycarbonylamino-propionic acid
methyl ester (43 mg, 0.145 mmol) were added and the resulting
reaction mixture was stirred at room temperature over the weekend.
The crude material was purified by flash chromatography on silica
gel using ethyl acetate and hexanes to afford 87 mg of title
compound. HRMS m/e 661.2237 (M+Na).sup.+
Preparation of
(S)-2-tert-butoxycarbonylamino-3-[4-(2,6-dichloro-4-hydroxy-benzoylamino)-
-phenyl]-propionic acid methyl ester
##STR00060##
[0165] To a solution of
(S)-2-tert-butoxycarbonylamino-3-[4-(2,6-dichloro-4-triisopropylsilanylox-
y-benzoylamino)-phenyl]-propionic acid methyl ester (84.7 mg, 0.132
mmol) in THF (1 mL) was added TBAF (199 .mu.L of 1 M solution in
THF) and the resulting mixture was stirred at room temperature
overnight. The solvent was removed under reduced pressure and the
residue, after redissolving in ethyl acetate, was washed with water
and brine and then dried over anhydrous sodium sulfate. The crude
material was purified by flash chromatography on silica gel using
ethyl acetate and hexanes to afford 50.3 mg of title compound. HRMS
m/e 505.0903 (M+Na).sup.+
Preparation of
(S)-2-amino-3-[4-(2,6-dichloro-4-hydroxy-benzoylamino)-phenyl]propionic
acid methyl ester hydrochloride
##STR00061##
[0167] To a solution of TMSCl (1.4 mL, 11.3 mmol) in MeOH (15 mL)
was added
(S)-2-tert-butoxycarbonylamino-3-[4-(2,6-dichloro-4-hydroxy-benzoyl-
amino)-phenyl]-propionic acid methyl ester (548 mg, 1.13 mmol) and
the resulting mixture was stirred at room temperature overnight.
The crude mixture was concentrated under reduced pressure and the
residue was triturated with diethyl ether to afford 379 mg of the
title compound. HRMS m/e 383.0561 (M+H).sup.+
Preparation of
N-[3-(tert-butyl-dimethyl-silanyloxy)-benzyl]-2-chloro-terephthalamic
acid methyl ester
##STR00062##
[0169] Preparation of 2-chloro-N-(3-hydroxy-benzyl)-terephthalamic
acid methyl ester is described in patent WO 01/58853.
2-Chloro-N-(3-hydroxy-benzyl)-terephthalamic acid methyl ester (4.0
g, 12.54 mmol), TBDMSCl (2.3 g, 15.0 mmol) and imidazole (1.9 g,
27.6 mmol) were dissolved in DMF (80 mL) and stirred at room
temperature overnight. Then the reaction mixture was diluted with
ethyl acetate, washed with water and brine and then dried over
anhydrous sodium sulfate. Crude material was purified by flash
chromatography on silica gel using ethyl acetate and hexanes to
afford 5.0 g of the title compound. MS m/e 433.9 (M+H).sup.+
Preparation of
N-[3-(tert-butyl-dimethyl-silanyloxy)-benzyl]-2-chloro-terephthalamic
acid
##STR00063##
[0171] To a solution of
N-[3-(tert-butyl-dimethyl-silanyloxy)-benzyl]-2-chloro-terephthalamic
acid methyl ester (4.9 g, 11.29 mmol) in 1,2-dichloroethane (80 mL)
was added trimethyltin hydroxide (20.4 g, 112.9 mmol) and the
resulting reaction mixture was stirred at 80.degree. C. for 8 h.
The solvent was removed under reduced pressure and the residue was
dissolved in ethyl acetate. It was then washed with an aqueous
solution of KHSO.sub.4, dried over anhydrous sodium sulfate and
filtered through the silica pad. The filtrate was concentrated
under reduced pressure to afford 4.0 g of the title compound. HRMS
m/e 420.1393 (M+H).sup.+
Preparation of
(S)-2-{4-[3-(tert-butyl-dimethyl-silanyloxy)-benzylcarbamoyl]-2-chloro-be-
nzoylamino}-3-[4-(2,6-dichloro-4-hydroxy-benzoylamino)-phenyl]-propionic
acid methyl ester
##STR00064##
[0173] To a solution of
N-[3-(tert-butyl-dimethyl-silanyloxy)-benzyl]-2-chloro-terephthalamic
acid (103 mg, 0.246 mmol) in DMF (2 mL) were added HBTU (103 mg,
0.271 mmol), DIEA (128 .mu.L, 0.738 mmol) and
(S)-2-amino-3-[4-(2,6-dichloro-4-hydroxy-benzoylamino)-phenyl]propionic
acid methyl ester hydrochloride salt (103 mg, 0.246 mmol). The
resulting reaction mixture was stirred at room temperature over the
weekend. Then it was diluted with ethyl acetate, washed with water
and brine. Crude material was purified by flash chromatography on
silica gel using methanol/methylene chloride to afford 100 mg of
the title compound. HRMS m/e 784.1776 (M+H).sup.+
Preparation of
(S)-3-{4-[4-(3-tert-butoxycarbonylamino-propoxy)-2,6-dichlorobenzoylamino-
]-phenyl}-2-[2-chloro-4-(3-hydroxy-benzylcarbamoyl)-benzoylamino]-propioni-
c acid methyl ester
##STR00065##
[0175] To a solution of
(S)-2-{4-[3-(tert-butyl-dimethyl-silanyloxy)-benzylcarbamoyl]-2-chloro-be-
nzoylamino}-3-[4-(2,
6-dichloro-4-hydroxy-benzoylamino)-phenyl]-propionic acid methyl
ester (91.5 mg, 0.116 mmol) in a mixture of acetone (1 mL) and DMF
(1 mL) were added potassium carbonate (48 mg, 3 eq.) and
(3-bromo-propyl)-carbamic acid tert-butyl ester (33 mg, 1.2 eq.).
The resulting reaction mixture was stirred at 75.degree. C.
overnight. Then it was diluted with ethyl acetate and washed with
water and brine and dried over anhydrous sodium sulfate. The crude
material was purified by flash chromatography on silica gel using
methanol/methylene chloride to afford 76.5 mg of the title
compound. HRMS m/e 827.2016 (M+H).sup.+
Preparation of
(S)-3-{4-[4-(3-amino-propoxy)-2,6-dichloro-benzoylamino]-phenyl}-[2-chlor-
o-4-(3-hydroxy-benzylcarbamoyl)-benzoylamino]-propionic acid methyl
ester hydrochloride
##STR00066##
[0177] Trimethylsilyl chloride (100 .mu.L, 10 eq.) was added to
methanol (2 mL). After 5 min the resulting solution was added to
(S)-3-{4-[4-(3-tert-butoxycarbonylamino-propoxy)-2,6-dichlorobenzoylamino-
]-phenyl}-2-[2-chloro-4-(3-hydroxy-benzylcarbamoyl)-benzoylamino]-propioni-
c acid methyl ester (65.6 mg, 0.079 mmol) and stirred at
temperature overnight. The crude reaction mixture was concentrated
and triturated with diethyl ether to afford 60.4 mg of the title
compound. HRMS m/e 727.1492 (M+H).sup.+
Preparation of
(S)-3-{4-[4-(3-Amino-propoxy)-2,6-dichloro-benzoylamino]-phenyl}-2-[2-chl-
oro-4-(3-hydroxy-benzylcarbamoyl)-benzoylamino]-propionic acid;
LFA-1 Ligand 4
##STR00067##
[0179] To a solution of
(S)-3-{4-[4-(3-amino-propoxy)-2,6-dichloro-benzoylamino]-phenyl}-[2-chlor-
o-4-(3-hydroxy-benzylcarbamoyl)-benzoylamino]-propionic acid methyl
ester hydrochloride (55 mg, 0.072 mmol) in methanol (1 mL) was
added an aqueous solution of 2M NaOH (178 .mu.L, 5 eq.). The
resulting reaction mixture was stirred at room temperature
overnight. Then it was neutralized with 1N HCl, lyophilized and
used for the next step without further purification. MS m/e 713.0
(M+H).sup.+
Example 1
Preparation of LFA-1 Ligand Reagent 1
##STR00068##
[0181] A solution of
(S)-3-amino-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimid-
ine-5-carbonyl}-amino)-propionic acid hydrochloride (0.114 mmol) in
acetonitrile (1 mL) and a solution of
succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol]ester
(58 mg, 0.114 mmol) in 1 mL of DMSO and diisopropylethylamine (40
.mu.L, 0.228 mmol). Both solutions were combined and stirred at
room temperature for 30 min. The crude reaction mixture was
concentrated under reduced pressure and purified by SFC to afford
51 mg of the title product. HRMS m/e 786.3665 (M+H).sup.+
Example 2
Preparation of LFA-1 Ligand Reagent 2
##STR00069##
[0183] The title compound was prepared in a similar manner with
(S)-3-amino-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimid-
ine-5-carbonyl}-amino)-propionic acid hydrochloride and
succinimidyl-[(N-maleimidopropionamido)-octaethyleneglycol]ester as
shown in Example 1. HRMS m/e 984.4530 (M+Na).sup.+
Example 3
Preparation of LFA-1 Ligand Reagent 3
##STR00070##
[0185] The title compound was prepared in a similar manner with
(S)-3-amino-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimid-
ine-5-carbonyl}-amino)-propionic acid hydrochloride and
succinimidyl-[(N-maleimidopropionamido)-dodecaethyleneglycol]ester
as shown in Example 1.
[0186] HRMS m/e 1138.5761 (M+H).sup.+
Example 4
Preparation of LFA-1 Ligand Reagent 4
##STR00071##
[0188] To a solution of
(S)-3-{4-[4-(3-amino-propoxy)-2,6-dichloro-benzoylamino]-phenyl}-2-[2-chl-
oro-4-(3-hydroxy-benzylcarbamoyl)-benzoylamino]-propionic acid
(0.196 mmol) in DMSO (2 mL) were added DIEA (102 .mu.L, 3 eq.) and
succinimidyl-[(N-maleimidopropionamido)-octaethyleneglycol]ester
(135 mg, 1 eq.). The resulting mixture was stirred at room
temperature for 1 h. Crude material was purified by HPLC to afford
105 mg of the title compound.
[0189] HRMS m/e 1287.4077 (M+H).sup.+
Example 5
Preparation of LFA-1 Ligand Reagent 5
##STR00072##
[0191] The title compound was prepared in a similar manner with
(S)-3-{4-[4-(3-amino-propoxy)-2,6-dichloro-benzoylamino]-phenyl}-2-[2-chl-
oro-4-(3-hydroxy-benzylcarbamoyl)-benzoylamino]-propionic acid and
succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol]ester
as shown in Example 4.
[0192] HRMS m/e 1111.3021 (M+H).sup.+
Example 6
Preparation of LFA-1 Ligand Reagent 6
##STR00073##
[0194] The title compound was prepared in a similar manner with of
(S)-3-{4-[4-(3-amino-propoxy)-2,6-dichloro-benzoylamino]-phenyl}-2-[2-chl-
oro-4-(3-hydroxy-benzylcarbamoyl)-benzoylamino]-propionic acid and
succinimidyl-[(N-maleimidopropionamido)-dodecaethyleneglycol]ester
as shown in Example 4.
[0195] HRMS m/e 732.2595 (M+2H).sup.2+
Example 7
Preparation of LFA-1 Ligand Reagent 7
##STR00074##
[0197] To a solution of
(S)-3-{4-[4-(3-amino-propoxy)-2,6-dichloro-benzoylamino]-phenyl}-2-[2-chl-
oro-4-(3-hydroxy-benzylcarbamoyl)-benzoylamino]-propionic acid
(0.131 mmol) in DMSO (2 mL) were added DIEA (46 .mu.L, 2 eq.) and
3-[2-(2-{2-[2-(2-{2-[2-(2-acetylsulfanyl-ethoxy)-ethoxy]-ethoxy}-ethoxy)--
ethoxy]-ethoxy}-ethoxy)-ethoxy]-propionic acid
2,5-dioxo-pyrrolidin-1-yl ester (78 mg, 1 eq.). The resulting
mixture was stirred at room temperature for 1 h. Crude material was
purified by HPLC to afford 114 mg of the title compound.
[0198] HRMS m/e 1195.3511 (M+H).sup.+
Example 8
Preparation of LFA-1 Ligand Reagent 8
##STR00075##
[0200] The title compound was prepared in a similar manner with
(S)-3-[3-(3-amino-propoxy)-benzoylamino]-2-({2-[3-(3-hydroxy-phenyl)-prop-
ylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid
and
succinimidyl-[(N-maleimidopropionamido)-octaethyleneglycol]ester as
shown in Example 1.
[0201] HRMS m/e 1139.5511 (M+H).sup.+
Example 9
Preparation of LFA-1 Ligand Reagent 9
##STR00076##
[0203] The title compound was prepared in a similar manner with
(S)-3-[3-(3-amino-propoxy)-5-hydroxy-benzoylamino]-2-({2-[3-(3-hydroxy-ph-
enyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic
acid and
succinimidyl-[(N-maleimidopropionamido)-octaethyleneglycol]ester as
shown in Example 1.
[0204] HRMS m/e 1155.5448 (M+H).sup.+
Example 10
Preparation of LFA-1 Ligand Reagent 10
##STR00077##
[0206] The title compound was prepared in a similar manner with
(S)-3-[3-(3-amino-propoxy)-5-hydroxy-benzoylamino]-2-({2-[3-(3-hydroxy-ph-
enyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic
acid and
succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol]este- r
as shown in Example 1.
[0207] HRMS m/e 979.4403 (M+H).sup.+
Preparation of Fluorescein (FITC) Labeled Targeting Reagents
[0208] The targeting reagents may be derivatized with fluorophores
that may be useful for studying their binding tracking to cells
that express receptors to the targeting small molecules. Such
molecules may be made in either or both of two methods. First, it
is possible to perform the reaction of the targeted maleimides with
2-[(5-fluoroseinyl)aminocarbonyl]ethylmercaptane. Alternatively,
the one-pot reaction of the integrin antagonist small molecule
targeting ligands, with
2-[(5-fluoroseinyl)aminocarbonyl]ethylmercaptane and the
bi-functional PEG reagent which is shown in Schemes 17 and 18.
[0209] Example of Method a)
Preparation of
(S)--N-[4-[3-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-(2,5-dioxo-2,5-dih-
ydro-pyrrol-1-yl)-propionylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy-
]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-propionylamino]propoxy]-phenyl-
]-3-[2-[3-(guanidino)-benzoylamino]-acetylamino]-succinamic
acid-FITC
##STR00078##
[0211] To a yellow suspension of
(S)--N-[4-[3-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-(2,5-dioxo-2,5-dih-
ydro-pyrrol-1-yl)-propionylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy-
]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-propionylamino]propoxy]-phenyl-
]-3-[2-[3-(guanidino)-benzoylamino]-acetylamino]-succinamic acid
(37.5 mg, 0.03 mmol) and
2-[(5-fluoroseinyl)aminocarbonyl]ethylmercaptane (FITC reagent)
(15.6 mg, 0.036 mml) in methanol (5 mL) was added an excess of
DIPEA (38.7 mg, 52 uL, 0.3 mmol) at room temperature under nitrogen
atmosphere. The resulting light yellow suspension was stirred for 2
h at which time LCMS analysis indicated the absence of starting
material. Then, the excess DIPEA was removed under vacuum and the
desired product was isolated by purification using HPLC to obtain
25 mg (50% yield) of
(S)--N-[4-[3-[3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[3-(2,5-dioxo-2,5-dih-
ydro-pyrrol-1-yl)-propionylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy-
]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]-propionylamino]propoxy]-phenyl-
]-3-[2-[3-(guanidino)-benzoylamino]-acetylamino]-succinamic
acid-FITC derivative as a brown solid.
[0212] ES(+)-HRMS m/e calcd. for C.sub.80H.sub.104N.sub.10O.sub.28S
(M+2H).sup.2+ 843.3444, obsd. 843.3437.
[0213] LCMS data=M+H, 1687.6
Example of Method b)
##STR00079##
[0215] Step 1.
[0216] Cystamine dihydrochloride (68 mg, 0.301 mmol) and DIEA (110
.mu.L, 2.1 eq.) were dissolved in DMF (10 mL), followed by addition
of NHS-fluorescein, a mixture of 5- and 6-carboxyfluorescein (300
mg, 0.634 mmol) and the resulting reaction mixture was stirred
overnight at room temperature. Then it was diluted with ethyl
acetate and washed three times with water and one time with brine.
The extract was dried over anhydrous sodium sulfate, concentrated
under reduced pressure, redissolved in small amount of methanol and
ethyl acetate, and then triturated with diethyl ether to obtain 140
mg of fluorescein-cystamine adduct as a bright orange solid.
[0217] Step 2.
[0218] The fluorescein-cystamine adduct (80 mg, 0.092 mmol) was
dissolved in a 3:1 mixture of methanol and water (4 mL) and TCEP
hydrochloride (80 mg, 3 eq.) was added. The resulting reaction
mixture was stirred at room temperature for 2 h. The product was
purified by HPLC to yield 78 mg of the product. LRMS (ESI)
435.0
Preparation of Fluorescein-Labeled Small Molecule-PEG
Conjugates
##STR00080##
[0220] General Procedure:
[0221] To a solution of ligand (1 eq.) in DMSO was added DIEA (2
eq.) and SM(PEG).sub.4n (1 eq.). The resulting reaction mixture was
stirred at room temperature for 1 h. Next, fluorescein with thiol
handle (1 eq.) was added and the reaction mixture was stirred for
an additional 10 min. The product was purified by HPLC.
[0222] Procedures for Covalent Attachment to Small Molecule
Integrin Targeting Ligands to 5'-thiol-siRNA Oligonucleotides siRNA
Preparation.
Oligoribonucleotide Synthesis
[0223] Oligoribonucleotides were synthesized according to the
phosphoramidite technology on solid phase employing an ABI 394
synthesizer (Applied Biosystems) at the 10 .mu.mol scale. For RNA
sequence information see tables 1 and 2. The corresponding siRNAs
are directed against the house keeping gene AHA1. Syntheses were
performed on a solid support made of controlled pore glass (CPG,
520 .ANG., with a loading of 75 .mu.mol/g, obtained from Prime
Synthesis, Aston, Pa., USA). Regular RNA phosphoramidites,
2'-O-Methylphosphoramidites as well as ancillary reagents were
purchased from Proligo (Hamburg, Germany). Specifically, the
following amidites were used:
(5'-O-dimethoxytrityl-N.sup.6-(benzoyl)-2'-O-t-butyldimethylsilyl-adenosi-
ne-3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite,
5'-O-dimethoxytrityl-N.sup.4-(acetyl)-2'-O-t-butyldimethylsilyl-cytidine--
3'-O-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite,
(5'-O-dimethoxytrityl-N.sup.2-(isobutyryl)-2'-O-t-butyldimethylsilyl-guan-
osine-3'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite, and
5'-O-dimethoxytrityl-2'-O-t-butyldimethylsilyl-uridine-3'-O-(2-cyanoethyl-
-N,N-diisopropylamino)phosphoramidite. 2'-O-Methylphosphoramidites
carried the same protecting groups as the regular RNA amidites. All
amidites were dissolved in anhydrous acetonitrile (100 mM) and
molecular sieves (3 .ANG.) were added. To generate the sulfhydryl
linker at the 5'-end of the oligomer the
1-O-Dimethoxytrityl-hexyl-disulfide,1'-[(2-cyanoethyl)-(N,N-diisopropyl)]-
-phosphoramidite linker from Glen Research (Sterling, Va., USA) was
used. Prior to small molecule conjugation the disulfide linker was
reduced using Tris-(2-carboxyethyl)phosphine (TCEP, see below). For
5'-end labeling with the Nu547 fluorophore the corresponding
phosphoramidite obtained from Thermo Fisher (Milwaukee, Wis.) was
employed. 5-Ethyl thiotetrazole (ETT, 500 mM in acetonitrile) was
used as activator solution. Coupling times were 6 minutes. In order
to introduce phosphorothioate linkages a 100 mM solution of
3-ethoxy-1,2,4-dithiazoline-5-one (EDITH, obtained from Link
Technologies, Lanarkshire, Scotland) in anhydrous acetonitrile was
employed.
Cleavage and Deprotection of Support Bound Oligomer
[0224] After finalization of the solid phase synthesis, the dried
solid support was transferred to a 15 mL tube and treated with
methylamine in methanol (2M, Aldrich) for 180 min at 45.degree. C.
After centrifugation the supernatant was transferred to a new 15 mL
tube and the CPG was washed with 1200 .mu.L
N-methylpyrrolidin-2-one (NMP, Fluka, Buchs, Switzerland). The
washing was combined with the methanolic methylamine solution and
450 .mu.L Triethylamine trihydrofluoride (TEA.3HF, Alfa Aesar,
Karlsruhe, Germany) was added. This mixture was brought to
65.degree. C. for 150 min. After cooling to room temperature 0.75
mL NMP and 1.5 mL of ethoxytrimethylsilane (Fluka, Buchs,
Switzerland) was added. 10 min later, the precipitated
oligoribonucleotide was collected by centrifugation, the
supernatant was discarded and the solid was reconstituted in 1 mL
buffer A (see below).
Purification of Oligoribonucleotides
[0225] Crude oligoribonucleotides were purified by strong anion
exchange (SAX) HPLC employing a preparative 22.times.250 mm DNA Pac
100 column (Dionex, Idstein, Germany) on an AKTA Explorer system
(GE Healthcare). Buffer A consisted of 10 mM NaClO.sub.4, 1 mM
EDTA, 10 mM Tris, pH 7.4, 6M Urea and 20% acetonitrile. Buffer B
had 500 mM NaClO.sub.4 in Buffer A. A flow rate of 4.5 mL/min was
employed. UV traces at 260 and 280 nm were recorded. A gradient of
20% B to 45% B within 55 min was employed. Appropriate fractions
were pooled and precipitated with 3M NaOAc, pH=5.2 and 70%
Ethanol.
[0226] Crude Nu547 labeled oligomers were purified by RP HPLC using
a XTerra Prep MS C8 10.times.50 mm column (Waters, Eschborn,
Germany) on an AKTA Explorer system (GE Helthcare). Buffer A was
100 mM triethylammonium acetate (Biosolve, Valkenswaard, The
Netherlands) and buffer B contained 50% acetonitrile in buffer A. A
flow rate of 5 mL/min was employed. UV traces at 260, 280 and 547
nm (in case of Nu547 labeled oligoribonucleotide) were recorded. A
gradient of 5% B to 60% B within 58 column volumes (CV) was
employed. Appropriate fractions were pooled and precipitated with
3M NaOAc, pH=5.2 and 70% Ethanol.
[0227] Finally, the purified oligomer was desalted by size
exclusion chromatography on a column containing Sephadex G-25 (GE
Healthcare). The concentration of the solution was determined by
absorbance measurement at 260 nm in a UV photometer (Beckman
Coulter, Krefeld, Germany). Until annealing the individual strands
were stored as frozen solutions at -20.degree. C.
Preparation of Small Molecule RNA Conjugates
[0228] Small molecules equipped with a maleimide functionality were
covalently conjugated to the RNA through a thioether linkage. To
enable this chemistry, .about.60 mg of the RNA containing the
5'-disulfide linker was reduced in water (5 mL) to the
corresponding thiol using 1 mL TCEP (0.5 M in water, obtained from
Sigma Aldrich). Once analytical anion exchange HPLC indicated
completion of the reaction (.about.2 h at room temperature) the RNA
was precipitated with 30 mL ethanol/3M NaOAc (pH 5.4) 32:1 (v/v)
over night at -20.degree. C. The pellet was collected by
centrifugation and used for the subsequent small molecule
conjugation.
[0229] In a typical conjugation reaction 10 mg RNA was dissolved in
2 mL sodium phosphate buffer (0.1 M, pH 7.0). To this solution the
small molecule (0.12 mM) in ACN/NMP 1:1 (v/v) was added over a
period of 5 minutes. Once RP LC-ESI MS showed consumption of the
input RNA the mixture was diluted with water (.about.10 mL) and
.about.40 mL ethanol/3M NaOAc (pH 5.4) 32:1 (v/v) was added to
precipitate the conjugated RNA over night at -20.degree. C. The
pellet was collected by centrifugation, dissolved in water and if
necessary purified by anion exchange HPLC pursuing the procedure
given above. If the conjugate is sufficiently pure the reaction
mixture was filtered through a size exclusion column (Sephadex
G-25, GE Healthcare).
Annealing of Oligoribonucleotides to Generate siRNA
[0230] Complementary strands were annealed by combining equimolar
RNA solutions. The mixture was lyophilized and reconstituted with
an appropriate volume of annealing buffer (100 mM NaCl, 20 mM
sodium phosphate, pH 6.8) to achieve the desired concentration.
This solution was placed into a water bath at 95.degree. C. which
was cooled to rt within 3 h. Table 3: siRNA sequence information;
lower case letters: 2'-OMe nucleotide; s: phosphorothioate linkage;
dT: deoxythymidine; (C6SSC6): C-6 disulfide linker; (Cy5): cyanine
5 dye.
[0231] The following assay was conducted to assess effect of
targeted molecules on the sLFA-1/ICAM-1 ELISA and Mac-1/ICAM-1
interactions.
[0232] Plates were coated with either 50 .mu.l/well of 2.0 ug/ml
solution of sLFA-1 or Mac-1 receptor in divalent cation buffer (1
mM MnCI.sub.2, 0.14M NaCl, 20 mM HEPES pH 7.2) at 4.degree. C.
overnight. Two hundred fifty .mu.l of blocking buffer (1% BSA in
divalent cation buffer) was added to each well 1 hour at 37.degree.
C. Plates were washed 3 times with wash buffer (TBS/0.05%
Tween-20/1 mM MnCl.sub.2). The compound to be tested was
solubilized in DMSO. A series of 1:3 dilutions were performed to
achieve a concentration range of 0.45 nM-3 uM. Fifty .mu.l of
binding buffer (0.5% BSA in divalent cation buffer)/1% DMSO and 50
.mu.l of the solutions to be tested were added to the appropriate
wells and incubated for 1 hour. Fifty .mu.l of 5dICAM-Fc (27 ng/ml)
was added to the appropriate wells and 50 .mu.l binding buffer was
added to non-specific binding wells and incubated for 2 hours and
washed. One hundred .mu.l of 1:4000 HRP-goat anti-huIgG was added
to each well and incubated for 1 hour and washed. One hundred .mu.l
of 1:1 TMB solution was added to each well and developed for 20 min
at room temperature. Color development was stopped by adding 100
.mu.l H.sub.3PO.sub.4 to each well. Absorbance was measured at 450
nm. These results are shown below in the Table 4 and 5.
[0233] The control compounds (142 and 143) were determined to have
an IC.sub.50 of about 37 and 11 nM respectively. The LFA-1
receptors of the cells were presumably bound to or associated with
the control compound.
##STR00081##
Evidence of Cellular Permeability and Localization of Small
Molecule Derivatives for Covalently Linked Integrin Antagonists to
FITC Fluorophores and siRNA for Targeted Delivery
Procedure
[0234] AML MV4-11 cells in growth medium (RPMI 1640 with 10% FBS)
were incubated with Duplex-27 (500 nM) for 1 hour at 37.degree. C.
For determining VLA-4 independent binding, 140 (10 .mu.M) was
included in one condition to block VLA-4 dependent binding. After
incubation, the cells were then washed twice with D-PBS and fixed
in 1% paraformaldehyde for 10 minutes. The uptake of siRNA was
analyzed by imaging flow cytometry using ImageStreamx (Aminis
Corporation, Seattle). The results are shown in Table A and in
FIGS. 1-4.
TABLE-US-00001 TABLE A Mean Cy3 Compound (concentration) intensity
Nothing 638 140 (10 .mu.M) 663 Duplex-27 (500 nM) 4007 140 (10
.mu.M) + Duplex-27 (500 nM) 2273
Assay of 5'-Sense Strand Modified siRNA for Knock-Down of AHA1 mRNA
in Cellular Systems
[0235] Materials and Methods
Reference gene: GAPDH Cell line: H1299_Nut-One Plating density:
5,000 cells/well Plating format: 96-well Time from plating to
treatment: 0 Control treatment: mock, untreated, control siRNA
Transfection reagent: DharmaFectl
Transfection Method Reverse TF
[0236] TF Reagent volume/well 0.15 mL siRNA final concentration 50
nM Assay method: Day 1 manual/Day 2 Washer
[0237] Reverse transfection: H1299 cells were transfected with
indicated siRNA at final concentration of 50 nM using DharmaFect-1
transfection reagent at 0.15 .mu.l/well. Cells were then plated
into 96-well plate at 5000 cells/well and incubated at 37.degree.
C. for 48 hours.
[0238] The efficacy of siRNA knock-down was measured with a
Branched DNA Assay as reported by the vendor; the results of such
knockdown are shown in FIG. 5. The relative cell viability was
assessed by the absolute expression of GAPDH in the same well (FIG.
6).
[0239] Unless stated to the contrary, all compounds in the examples
were prepared and characterized as described. All patents and
publications cited herein are hereby incorporated by reference in
their entirety.
TABLE-US-00002 TABLE 1 Summaries of the composition of
5'-derivatized siRNA single and double strands Duplex-ID Sense-ID
Sequence 5'-->3' Antisense-ID Sequence 5'-->3' Duplex-1
Sense-1 GGAuGAAGuGGAGAuuAGudTsdT Antisense-1
ACuAAUCUCcACUUcAUCCdTsdT Duplex-2 Sense-2 LFA-1 Ligand Reagent 1-
Antisense-1 ACuAAUCUCcACUUcAUCCdTsdT (SC6)GGAuGAAGuGGAGAuuAGudTsdT
Duplex-3 Sense-3 LFA-1 Ligand Reagent 3- Antisense-1
ACuAAUCUCcACUUcAUCCdTsdT (SC6)GGAuGAAGuGGAGAuuAGudTsdT Duplex-8
Sense-8 LFA-1 Ligand Reagent 6- Antisense-1
ACuAAUCUCcACUUcAUCCdTsdT (SC6)GGAuGAAGuGGAGAuuAGudTsdT Duplex-9
Sense-9 LFA-1 Ligand Reagent 5- Antisense-1
ACuAAUCUCcACUUcAUCCdTsdT (SC6)GGAuGAAGuGGAGAuuAGudTsdT Duplex-10
Sense-10 LFA-1 Ligand Reagent 4- Antisense-1
ACuAAUCUCcACUUcAUCCdTsdT (SC6)GGAuGAAGuGGAGAuuAGudTsdT Duplex-19
Sense-19 LFA-1 Ligand Reagent 8- Antisense-1
ACuAAUCUCcACUUcAUCCdTsdT (SC6)GGAuGAAGuGGAGAuuAGudTsdT Duplex-20
Sense-20 LFA-1 Ligand Reagent 9- Antisense-1
ACuAAUCUCcACUUcAUCCdTsdT (SC6)GGAuGAAGuGGAGAuuAGudTsdT Duplex-21
Sense-21 LFA-1 Ligand Reagent 10- Antisense-1
ACuAAUCUCcACUUcAUCCdTsdT (SC6)GGAuGAAGuGGAGAuuAGudTsdT
TABLE-US-00003 TABLE 2 Analytical Data for small molecule siRNA
conjugates IEX Small Tar- Calc. Exp. % Molecule get Number Sequence
(5'--3') Mass Mass FLP LFA-1-PEG4- Aha1 Sense-2 LFA-1 Ligand
Reagent 1:- 7882.13 7884.8 86 maleimide
(SC6)GGAuGAAGuGGAGAuuAGudTsdT LFA-1-PEG12- Aha1 Sense-3 LFA-1
Ligand Reagent 3- 8233.84 8237.5 88.8 maleimide
(SC6)GGAuGAAGuGGAGAuuAGudTsdT LFA-1-PEG12- Aha1 Sense-8 LFA-1
Ligand Reagent 6- 8562.13 8562 90.9 maleimide
(SC6)GGAuGAAGuGGAGAuuAGudTsdT LFA-1-PEG4- Aha1 Sense-9 LFA-1 Ligand
Reagent 5- 8209.7 8210 92.9 maleimide (SC6)GGAuGAAGuGGAGAuuAGudTsdT
LFA-1-PEG8- Aha1 Sense-10 LFA-1 Ligand Reagent 4- 8385.92 8386 93.4
maleimide (SC6)GGAuGAAGuGGAGAuuAGudTsdT
TABLE-US-00004 TABLE 3 Summary of siRNA sequences where in the
5'-antisense strand has been derivatized with Nu547 Sense-
Duplex-ID ID Sequence 5'-->3' Antisense-ID Sequence 5'-->3'
Duplex-22 Sense- LFA-1 Ligand Reagent 1:- Antisense-1
(Nu547)ACuAAUCUCcACUUcAUCCdTsdT 22 (SC6)GGAuGAAGuGGAGAuuAGudTsdT
Duplex-23 Sense- LFA-1 Ligand Reagent 3- Antisense-1
(Nu547)ACuAAUCUCcACUUcAUCCdTsdT 23 (SC6)GGAuGAAGuGGAGAuuAGudTsdT
Duplex-28 Sense- LFA-1 Ligand Reagent 6- Antisense-1
(Nu547)ACuAAUCUCcACUUcAUCCdTsdT 28 (SC6)GGAuGAAGuGGAGAuuAGudTsdT
Duplex-29 Sense- LFA-1 Ligand Reagent 5- Antisense-1
(Nu547)ACuAAUCUCcACUUcAUCCdTsdT 29 (SC6)GGAuGAAGuGGAGAuuAGudTsdT
Duplex-30 Sense- LFA-1 Ligand Reagent 4- Antisense-1
(Nu547)ACuAAUCUCcACUUcAUCCdTsdT 30 (SC6)GGAuGAAGuGGAGAuuAGudTsdT
Duplex-35 Sense- LFA-1 Ligand Reagent 8- Antisense-1
(Nu547)ACuAAUCUCcACUUcAUCCdTsdT 35 (SC6)GGAuGAAGuGGAGAuuAGudTsdT
Duplex-36 Sense- LFA-1 Ligand Reagent 9- Antisense-1
(Nu547)ACuAAUCUCcACUUcAUCCdTsdT 36 (SC6)GGAuGAAGuGGAGAuuAGudTsdT
Duplex-37 Sense- LFA-1 Ligand Reagent 10- Antisense-1
(Nu547)ACuAAUCUCcACUUcAUCCdTsdT 37
(SC6)GGAuGAAGuGGAGAuuAGudTsdT
TABLE-US-00005 TABLE 4 Summary of small molecule-siRNA conjugate
potencies in integrin antagonists assays and siRNA KD data Jurkat
Configuration Cells/VCAM-1 .alpha.V.beta.3 LFA1 Targeting Small
molecule Ligand siRNA Adhesion Adhesion Adhesion Element Reagent
siRNA Fluorochrome derivative Assay (nM) Assay (nM) Assay (nM) AHA1
% KD None AHA-1 Duplex-1 No RD-03518 > 200 98 None AHA-1
Duplex-1N Nu547 RD-05170 > 200 LFA-1 Ligand LFA-1 Ligand AHA-1
Duplex-2 No RD-04705 870 97 1-PEG 4 Reagent 1 LFA-1 Ligand LFA-1
Ligand AHA-1 Duplex-3 No RD-04706 > 200 1090 97 1-PEG 12 Reagent
3 LFA-1 Ligand LFA-1 Ligand AHA-1 Duplex-22 Nu547 RD-04712 700 4
1-PEG 4 Reagent 1: LFA-1 Ligand LFA-1 Ligand AHA-1 Duplex-23 Nu547
RD-04713 634 14 1-PEG 4 Reagent 1: LFA-1 Ligand LFA-1 Ligand AHA-1
Duplex-6 No RD-04809 12 192 4-PEG 12 Reagent 6 LFA-1 Ligand LFA-1
Ligand AHA-1 Duplex-7 No RD-04810 5 141 4-PEG 4 Reagent 5 LFA-1
Ligand LFA-1 Ligand AHA-1 Duplex-10 No RD-04811 25 80 4-PEG 8
Reagent 4 LFA-1 Ligand LFA-1 Ligand AHA-1 Duplex-28 Nu547 RD-04819
13 204 4-PEG 12 Reagent 6 LFA-1 Ligand LFA-1 Ligand AHA-1 Duplex-29
Nu547 RD-04820 18 117 4-PEG 4 Reagent 5 LFA-1 Ligand LFA-1 Ligand
AHA-1 Duplex-31 Nu547 RD-04821 1 133 4-PEG 8 Reagent 4 LFA-1 Ligand
LFA-1 Ligand AHA-1 Duplex-19 No RD-05382 575 2-PEG 8 Reagent 8
LFA-1 Ligand LFA-1 Ligand AHA-1 Duplex-20 No RD-05383 391 3-PEG 8
Reagent 9 LFA-1 Ligand LFA-1 Ligand AHA-1 Duplex-21 No RD-05384 373
3-PEG 4 Reagent 10 LFA-1 Ligand LFA-1 Ligand AHA-1 Duplex-35 Nu547
RD-05425 532 2-PEG 8 Reagent 8 LFA-1 Ligand LFA-1 Ligand AHA-1
Duplex-36 Nu547 RD-05426 256 3-PEG 8 Reagent 9 LFA-1 Ligand LFA-1
Ligand AHA-1 Duplex-37 Nu547 RD-05427 324 3-PEG 4 Reagent 10 LFA-1
142 37 small molecule LFA-1 143 11 small molecule Negative assay
FITC-22 > 200 reference Negative assay FITC-23 > 200
reference
TABLE-US-00006 TABLE 5 Identity, characterization and binding
potencies of FITC isomer labeled reagents ##STR00082## ##STR00083##
Jurkat Cells/ Synthesis method: VCAM-1 LFA1 one pot or with
Adhesion .alpha.V.beta.3 Adhesion corresponding Assay Assay Assay
Observed Example Targeting Element targeting example (IC50 nM)
(IC50 nM) (IC50 nM) Calc. Mass Mass FITC-1 LFA-1 Ligand Method B
(one pot) 502 1138.27 1138.5761 1-PEG12 (M + H)+ FITC-2 LFA-1
Ligand Method B (one pot) 134 786.3669 786.3665 1-1-PEG4 [M + H]+
FITC-3 LFA-1 Ligand Method B (one pot) 500 500 795.87 796.3357
1-PEG4-FITC [M + 2H]2+ FITC-4 LFA-1 Ligand Method B (one pot) 160
160 1397.51 1397.5496 1-PEG8-FITC [M + H]+ FITC-5 LFA-1 Ligand
Method B (one pot) 340 1221.3 1221.4445 1-PEG4-FITC [M + H]+ FITC-6
LFA-1 Ligand Method B (one pot) 790 796.365 796.8230 2-PEG8-opened
[M + 2H]2+ maleimide-FITC FITC-7 LFA-1 Ligand Method B (one pot)
430 707.25 707.7625 3-PEG4- [M + 2H]2+ maleimide-FITC FITC-8 LFA-1
Ligand Method B (one pot) 380 804.365 804.8212 3-PEG8-opened [M +
2H]2+ maleimide-FITC FITC-9 LFA-1 Ligand Method B (one pot) 15
1900.28 861.7457 4-PEG12-FITC [M + 2H]2+ FITC-10 LFA-1 Ligand
Method B (one pot) 16 113 782.94 782.6987 4-PEG4-FITC [M + 2H]2+
FITC-11 LFA-1 Ligand Method B (one pot) 9 43 862.035 861.7459
4-PEG8-FITC [M + 2H]2+ FITC-22 untargeted benzyl- Method B (one
pot) 9,400 941.019 941.3269 PEG4-FITC (M + H)+ FITC-23 untargeted
benzyl- Method B (one pot) >200 >10,000 1117.23 1117.4317
PEG8-FITC [M + H]+ 142 positive control 74 140 positive control 4
141 positive control 2
* * * * *