Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
IMPROVED GASTRIN RELEASING PEPTIDE COMPOUNDS
CROSS REFERENCE TO RELATED APPLICATIONS
[0001 ] This application claims benefit of U.S. Application No. 10/828,925
filed April
20, 2004, which is a continuation-in-part application of International
Application
PCT/US03/041328, filed December 24, 2003, which claims priority to U.S.
Application No.
10/341,577 filed January 13, 2003. All of these applications are hereby
incorporated by
reference in their entirety.
FIELD OF THE INVENTION
[0002] This invention relates to novel gastrin releasing peptide (GRP)
compounds
which are useful as diagnostic imaging agents or radiotherapeutic agents.
These GRP
compounds are labeled with radionuclides or labels detectable by in vivo light
imaging and
include the use of novel linkers between the label and the targeting peptide,
which provides
for improved pharmacokinetics.
BACKGROUND OF THE INVENTION
[0003] The use of radiopharmaceuticals (e.g., diagnostic imaging agents,
radiotherapeutic agents) to detect and treat cancer is well known. In more
recent years, the
discovery of site-directed radiopharmaceuticals for cancer detection and/or
treatment has
gained popularity and continues to grow as the medical profession better
appreciates the
specificity, efficacy and utility of such compounds.
[0004] These newer radiopharmaceutical agents typically consist of a targeting
agent
connected to a metal chelator, which can be chelated to (e.g., complexed with)
a diagnostic
metal radionuclide such as, for example, technetium or indium, or a
therapeutic metal
radionuclide such as, for example, lutetium, yttrium, or rhenium. The role of
the metal
chelator is to hold (i.e., chelate) the metal radionuclide as the
radiopharmaceutical agent is
delivered to the desired site. A metal chelator which does not bind strongly
to the metal
radionuclide would render the radiopharmaceutical agent ineffective for its
desired use since
the metal radionuclide would therefore not reach its desired site. Thus,
further research and
development led to the discovery of metal chelators, such as that reported in
U.S. Pat. No.
5,662,885 to Pollak et. al., hereby incorporated by reference, which exhibited
strong binding
affinity for metal radionuclides and the ability to conjugate with the
targeting agent.
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Subsequently, the concept of using a "spacer" to create a physical separation
between the
metal chelator and the targeting agent was further introduced, fox example in
U.S. Pat.
5,976,495 to Pollak et. al., hereby incorporated by reference.
[0005] The role of the targeting agent, by virtue of its affinity for certain
binding
sites, is to direct the diagnostic agent, such as a radiopharmaceutical agent
containing the
metal radionuclide, to the desired site for detection or treatment. Typically,
the targeting
agent may include a protein, a peptide, or other macromolecule which exhibits
a specifac
affinity for a given receptor. Other known targeting agents include monoclonal
antibodies
(MAbs), antibody fragments (Fab's and (Fab)2's), and receptor-avid peptides.
Donald J.
Buchsbaum, "Cancer Therapy with Radiolabeled Antibodies; Pharmacokinetics of
Antibodies and Their Radiolabels; Experimental Radioimmunotherapy and Methods
to
Increase Therapeutic Efficacy," CRC Press, Boca Raton, Chapter 10, pp. 115-
140, (1995);
Fischman, et al. "A Ticket to Ride: Peptide Radiopharmaceuticals," The Journal
of Nuclear
Medicine, vol. 34, No. 12, (Dec. 1993). These references are hereby
incorporated by
reference in their entirety.
[0006] In recent years, it has been learned that some cancer cells contain
gastrin
releasing peptide (GRP) receptors (GRP-R) of which there are a number of
subtypes. In
particular, it has been shown that several types of cancer cells have over-
expressed or
uniquely expressed GRP receptors. For this reason, much research and study
have been done
on GRP and GRP analogues which bind to the GRP receptor family. One such
analogue is
bombesin (BBN), a 14 amino acid peptide (i.e., tetradecapeptide) isolated from
frog skin
which is an analogue of human GRP and which binds to GRP receptors with high
specificity
and with an affinity similar to GRP.
[0007] Bombesin and GRP analogues may take the form of agonists or
antagonists.
Binding of GRP or BBN agonists to the GRP receptor increases the rate of cell
division of
these cancer cells and such agonists are internalized by the cell, while
binding of GRP or
BBN antagonists generally does not result in either internalization by the
cell or increased
rates of cell division. Such antagonists are designed to competitively inhibit
endogenous
GRP binding to GRP receptors and reduce the rate of cancer cell proliferation.
See, e.g.,
Hoffken, K.; Peptides in Oncology II, Somatostatin Analogues and Bombesin
Antagonists
(1993), pp. S7-112. For this reason, a great deal of work has been, and is
being pursued to
develop BBN or GRP analogues that are antagonists. E.g., Davis et al.,
Metabolic Stability
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and Tumor Inhibition of Bombesin/GRP Receptor Antagonists, Peptides, vol. 13,
pp. 401-
407, 1992.
[0008] In designing an effective compound for use as a diagnostic or
therapeutic
agent for cancer, it is important that the drug have appropriate in vivo
targeting and
pharmacokinetic properties. For example, it is preferable that fox a
radiopharmaceutical, the
radiolabeled peptide have high specific uptake by the cancer cells (e.g., via
GRP receptors).
In addition, it is also preferred that once the radionuclide localizes at a
cancer site, it remains
there for a desired amount of time to deliver a highly localized radiation
dose to the site.
[0009] Moreover, developing radiolabeled peptides that are cleared efficiently
from
normal tissues is also an important factor for radiophannaceutical agents.
When
biomolecules (e.g., MAb, Fab or peptides) labeled with metallic radionuclides
(via a chelate
conjugation), are administered to an animal such as a human, a large
percentage of the
metallic radionuclide (in some chemical form) can become "trapped" in either
the kidney or
liver parenchyma (i.e., is not excreted into the urine or bile). Duncan et
al.; Indium-111-
Diethylenetriaminepentaacetic Acid-Octreotide Is Delivered in Vivo to
Pancreatic, Tumor
Cell, Renal, and Hepatocyte Lysosomes, Cancer Research 57, pp. 659-671, (Feb.
15, 1997).
For the smaller radiolabeled biomolecules (i.e., peptides or Fab), the major
route of clearance
of activity is through the kidneys which can also retain high levels of the
radioactive metal
(i.e., normally >10-15% of the injected dose). Retention of metal
radionuclides in the kidney
or liver is clearly undesirable. Conversely, clearance of the
radiopharmaceutical from the
blood stream too quickly by the kidney is also undesirable if longer
diagnostic imaging or
high tumor uptake for radiotherapy is needed.
[0010] Subsequent work, such as that in U.S. Pat. 6,200,546 and US
200210054855 to
Hoffinan, et. al, hereby incorporated by reference in their entirety, have
attempted to
overcome this problem by forming a compound having the general formula X-Y-B
wherein
X is a group capable of complexing a metal, Y is a covalent bond on a spacer
group and B is
a bombesin agonist binding moiety. Such compounds were reported to have high
binding
affinities to GRP receptors, and the radioactivity was retained inside of the
cells for extended
time periods. In addition, in vivo studies in normal mice have shown that
retention of the
radioactive metal in the kidneys was lower than that known in the art, with
the majority of the
radioactivity excreted into the urine.
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4
[0011 J New and improved radiopharmaceutical and other diagnostic compounds
which have improved pharmacokinetics and improved kidney excretion (i.e.,
lower retention
of the radioactive metal in the kidney) have now been found for diagnostic
imaging and
therapeutic uses. For diagnostic imaging, rapid renal excretion and low
retained levels of
radioactivity are critical for improved images. For radiotherapeutic use,
slower blood
clearance to allow for higher tumor uptake and better tumor targeting with low
kidney
retention are critical.
SUMMARY OF THE INVENTION
[0012] In an embodiment of the present invention, there is provided new and
improved compounds for use in diagnostic imaging or radiotherapy. The
compounds include
a chemical moiety capable of complexing a medically useful metal ion or
radionuclide (metal
chelator) attached to a GRP receptor targeting peptide by a linker or spacer
group. In another
embodiment, these compounds include an optical label (e.g. a photolabel or
other label
detectable by light imaging, optoacoustical imaging or photoluminescence)
attached to a GRP
receptor targeting peptide by a linker or spacer group.
[0013] In general, compounds of the present invention may have the formula:
M-N-O-P-G
wherein M is the metal chelator (in the form complexed with a metal
radionuclide or not), or
the optical label, N-O-P is the linker, and G is the GRP receptor targeting
peptide.
[0014] The metal chelator M may be any of the metal chelators known in the art
for
complexing with a medically useful metal ion or radionuclide. Preferred
chelators include
DTPA, DOTA, D03A, HP-D03A, EDTA, TETA, EHPG, HBED, NOTA, DOTMA,
TETMA, PDTA, TTHA, LICAM, MECAM, or peptide chelators, such as, for example,
those
discussed herein. The metal chelator may or may not be complexed with a metal
radionuclide, and may include an optional spacer such as a single amino acid.
Preferred
metal radionuclides for scintigraphy or radiotherapy include 99"'Tc, SICr,
67Ga, 68Ga, 47Sc,
SICr 167Tm 141Ce 111In lbs~,-b 175 140La 90y 88Y 153sm 166H~ 165Dy 166Dy 62Cua
64Cu,
> > > > > > > > > > >
67Cu 97Ru lo3Ru ls6Re lssRe Zo3Pb 211Bi 212Bi 213Bi 214Bi 225AC 105 lo9Pd
117~nSn
> > > > > > > > > > > > > >
149Pm, 161Tb, 177Lu, lg$Au and 199Au. The choice of metal will be determined
based on the
desired therapeutic or diagnostic application. For example, for diagnostic
purposes the
preferred radionuclides include 64Cu, 67Ga, 68Ga, 99mTc, and 111In, with
99mTc, and '''In being
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particularly preferred. For therapeutic purposes, the preferred radionuclides
include 64Cu,
9°~,' IOSRh, 111In' 117msn' 149Pm' 153Sm' 161.Lb' 166Dy' 166H~' 175'
177L.u' 186/188Re, and 199Au,
with 177Lu and 9°Y being particularly preferred. A most preferred
chelator used in
compounds ofthe invention is 1-substituted 4,7,10-tricarboxymethyl 1,4,7,10
5 tetraazacyclododecane triacetic acid (D03A).
[0015] The optical Label M may be any of various optical labels known in the
art.
Preferred labels include, without limitation, optical dyes, including organic
chromophores or
fluorophores, such as cyanine dyes light absorbing compounds, light reflecting
and scattering
compounds, and bioluminescent molecules.
[0016] In one embodiment, the linker N-O-P contains at least one non-alpha
amino
acid.
[0017] In another embodiment, the linker N-O-P contains at least one
substituted bile
acid.
[0018] In yet another embodiment, the linker N-O-P contains at least one non-
alpha
amino acid With a cyclic group.
[0019] The GRP receptor targeting peptide may be GRP, bombesin or any
derivatives
or analogues thereof. In a preferred embodiment, the GRP receptor targeting
peptide is a
GRP or bombesin analogue which acts as an agonist. In a particularly preferred
embodiment,
the GRP receptor targeting peptide is a bombesin agonist binding moiety
disclosed in U.S.
Pat. 6,200,546 and US 2002/005455, incorporated herein by reference.
[0020] There is also provided a novel method of imaging using the compounds of
the
present invention.
[0021 ] A single or mufti-vial kit that contains all of the components needed
to prepare
the diagnostic or therapeutic agents of the invention is provided in an
exemplary embodiment
of the present invention.
[0022] There is further provided a novel method for preparing a diagnostic
imaging
agent comprising the step of adding to an injectable imaging medium a
substance containing
the compounds of the present invention.
[0023] A novel method of radiotherapy using the compounds of the invention is
also
provided, as is a novel method for preparing a radiotherapeutic agent
comprising the step of
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adding to an injectable therapeutic medium a substance comprising a compound
of the
W vention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1A is a graphical representation of a series of chemical reactions
for the
synthesis of intermediate C ((3J3, 5/~)-3-(9H Fluoren-9-ylmethoxy)aminocholan-
24-oic acid),
from A (Methyl-(3J3, Sf3)-3-aminocholan-24-ate) and B ((3f3, SJ3)-3-
aminocholan-24-oic acid),
as described in Example I.
[0025] FIG. 1B is a graphical representation of the sequential reaction for
the
synthesis ofN [(313,513)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-
tetraazacyclododec-1-
yl]acetyl]amino] acetyl]amino] cholan-24-yl]-L-glutaminyl-L-tryptophyl-L-
alanyl-L-valyl-
glycyl-L-histidyl-L-leucyl-L-methioninamide (L62), as described in Example I.
[0026] FIG. 2A is a graphical representation of the sequential reaction for
the
synthesis ofN [4-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-
yl]acetyl]amino]acetyl]amino]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-
valyl-glycyl-
L-histidyl-L-leucyl-L-methioninamide (L70), as described in Example II.
[0027] FIG. 2B is a general graphical representation of the sequential
reaction for the
synthesis ofN [4-[2-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-
1-
y1] acetyl] amino] ethoxy]benzoyl] -L-glutaminyl-L-tryptophyl-L-al anyl-L-
valyl-glycyl-L-
histidyl-L-leucyl-L-methioninamide (L73), N [3-[[[[4,7,10-Tris(carboxymethyl)-
1,4,7,10-
tetraazacyclododec-1-yl]acetyl]amino]methyl]benzoyl]-L-glutaminyl-L-tryptophyl-
L-alany1-
L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L115), and N [4-
[[[[4,7,10-
Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]
acetyl]amino]methyl]phenylacetyl]-L-
glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-
methioninamide
(L116), as described in Example II.
[0028] FIG. 2C is a chemical structure of the linker used in the synthesis
reaction of
FIG. 2B for synthesis ofN [4-[2-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-
tetraazacyclododec-
1-yl] acetyl ] amino] ethoxy]benzoyl ]-L-glutaminyl-L-tryptophyl-L-al anyl-L-
val yl-glycyl-L-
histidyl-L-leucyl-L-methioninamide (L73), as described in Example II.
[0029] FIG. 2D is a chemical structure of the linker used in the synthesis
reaction of
FIG. 2B for synthesis ofN [3-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-
tetraazacyclododec-1-
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y1) acetyl) amino)methyl)benzoylJ-L-glutaminyl-L-tryptophyl-L-al any!-L-valyl-
glycyl-L-
histidyl-L-leucyl-L-methioninamide (L115), as described in Example II.
[0030) FIG. 2E is a chemical structure of the linker used in the synthesis
reaction of
FIG. 2B for synthesis ofN [4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-
tetraazacyclododec-1-
ylJacetyl)amino)methyl)phenylacetyl)-L-glutaminyl-L-tryptophyl-L-alanyl-L-
valyl-glycyl-L-
histidyl-L-leucyl-L-methioninamide (L116), as described in Example II.
[0031 ) FIG. 2F is a graphical representation of the sequential reaction for
the
synthesis ofN [[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-
yl)acetyl)glycyl-
4-piperidinecarbonyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-
histidyl-L-leucyl-
L-methioninamide (L74), as described in Example II.
[0032) FIG. 3A is a graphical representation of a series of chemical reactions
for the
synthesis of intermediate (313,513)-3-[[(9H Fluoren-9-
ylmethoxy)amino)acetyl)amino-12-
oxocholan-24-oic acid (C), as described in Example III.
[0033] FIG. 3B is a graphical representation of the sequential reaction for
the
synthesis ofN [(313,5J3)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-
tetraazacyclododec-1-
yl)acetylJaminoJacetyl]aminoJ-12,24-dioxocholan-24-ylJ-L-glutaminyl-L-
tryptophyl-L-
alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L67), as described
in Example
III.
[0034) FIG. 3C is a chemical structure of (313,513)-3-Amino-12-oxocholan-24-
oic acid
(B), as described in Example III.
[0035) FIG. 3D is a chemical structure of (313,513)-3-[[(9H Fluoren-9-
ylmethoxy)amino)acetyl)amino-12-oxocholan-24-oic acid (C), as described in
Example III.
[0036) FIG 3E is a chemical structure ofN-[(313,5f3)-3-[[[[[4,7,10-
Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-
ylJacetylJamino)acetyl)amino)-12,24-
dioxocholan-24-ylJ-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-
histidyl-L-leucyl-
L-methioninamide (L67), as described in Example III.
[0037) FIG. 4A is a graphical representation of a sequence of reactions to
obtain
intermediates (313,513,12a)-3-[[(9H-Fluoren-9-ylmethoxy)amino)acetyl)amino-12-
hydroxycholan-24-oic acid (3a) and (313,513,7a,12a)-3-[[(9H-Fluoren-9-
ylmethoxy)amino]acetyl)amino-7,12-dihydroxycholan-24-oic acid (3b), as
described in
Example IV.
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[0038] FIG. 4B is a graphical representation of the sequential reaction for
the
synthesis ofN [(313,513,12a)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-
tetraazacyclododec-
1-yl] acetyl] amino] acetyl] amino]-12-hydroxy-24-oxocholan-24-yl]-L-
glutaminyl-L-
tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L63),
as
described in Example IV.
[0039] FIG. 4C is a graphical representation of the sequential reaction for
the
synthesis ofN [(313,513,7a,12a)-3-[[[[(4,7,10-Tris(carboxymethyl)-1,4,7,10-
tetraazacyclo
dodec-1-yl] acetyl] amino] acetyl] amino]-7,12-dihydroxy-24-oxocholan-24-yl]-L-
glutaminyl-
L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamidc
(L64), as
described in Example IV.
[0040] FIG. 4D is a chemical structure of (313,513,7a,12a)-3-amino-7,12-
dihydroxycholan-24-oic acid (2b), as described in Example IV.
[0041] FIG. 4E is a chemical structure of (313,513,12a)-3-[[(9H-Fluoren-9-
ylmethoxy)amino]acetyl]amino-12-hydroxycholan-24-oic acid (3a), as described
in Example
IV;
[0042] FIG. 4F is a chemical structure of (313,513,7a,12a)-3-[[(9H-Fluoren-9-
ylmethoxy)amino]acetyl]amino-7,12-dihydroxycholan-24-oic acid (3b), as
described in
Example IV.
[0043] FIG. 4G is a chemical structure ofN [(313,513,12a)-3-[[[[[4,7,10-
Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-
yl]acetyl]amino]acetyl]amino]-12-
hydroxy-24-oxocholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-
L-histidyl-
L-leucyl-L-methioninamide (L63), as described in Example IV.
[0044] FIG. 4H is a chemical structure ofN ((313,513,7a,12a)-3-[[[[[4,7,10-
Tris(carboxymethyl)-1,4,7,10-tetraazacyclo dodec-1-
yl]acetyl]amino]acetyl]amino]-7,12-
dihydroxy-24-oxocholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-
glycyl-L-
histidyl-L-leucyl-L-methioninamide (L64), as described in Example IV.
[0045] FIG. 5A is a general graphical representation of the sequential
reaction for the
synthesis of4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-
y1] acetyl] amino]methyl]b enzoyl-L-glutaminyl-L-tryptophyl-L-al anyl-L-valyl-
glycyl-L
histidyl-L-leucyl-L-methioninamide (L71); and Trans-4-[[[[4,7,10-
tris(carboxymethyl)
1,4,7,10-tetraazacyclododec-1-yl] acetyl]amino]methyl] cyclohexylcarbonyl-L-
glutaminyl-L
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tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L72)
as described
in Example V, wherein the linker is from Fig. 5B and Fig. 5C, respectively.
[0046] FIG. 5B is a chemical structure of the linker used in compound L71 as
shown
in Fig. 5A and as described in Example V.
[0047] FIG. SC is a chemical structure of the linker used in compound L72 as
shown
in Fig. 5A and as described in Example V.
[0048] FIG. SD is a chemical structure of Rink amide resin functionalised with
bombesin[7-14] (B), as described in Example V.
[0049) FIG. SE is a chemical structure of Tf~an,r-4-[[[(9H-fluoren-9-
ylmethoxy)carbonyl]amino]methyl]cyclohexanecarboxylic acid (D), as described
in Example
V;
[0050] FIG. 6A is a graphical representation of a sequence of reactions for
the
synthesis of intermediate linker 2-[[[9H Fluoren-9-
ylmethoxy)carbonyl]amino]methyl]benzoic acid (E), as described in Example VI.
[0051] FIG. 6B is a graphical representation of a sequence of reactions for
the
synthesis of intermediate linker 4-[[[9H Fluoren-9-
ylmethoxy}carbonyl]amino]methyl]-3-
nitrobenzoic acid (H), as described in Example VI.
[0052] FIG. 6C is a graphical representation of the synthesis ofN [2-
[[[[4,7,10-
Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-
yl]acetyl]amino]methyl]benzoyl]-L-
glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-Ieucyl-L-
methioninamide
(L75), as described in Example VI.
[0053] FIG. 6D is a graphical representation of the synthesis of N [4-
[[[[4,7,10-
Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl] acetyl) amino]methyl]-3-
nitrobenzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-
leucyl-L-
methioninamide (L76), as described in Example VI.
[0054] FIG. 7A is a graphical representation of a sequence of reactions for
the
synthesis of intermediate linker [4-[[[9H Fluoren-9-
ylmethoxy)carbonyl]amino]methyl]phenoxy]acetic acid (E), as described in
Example VII.
[0055] FIG. 7B is a graphical representation of the synthesis of N [[4-
[[[[4,7,10-
Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-
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y1] acetyl] aminoJmethylJphenoxy] acetylJ-L-glutaminyl-L-tryptophyl-L-al anyl-
L-valyl-glycyl-
L-histidyl-L-leucyl-L-methioninamide (L124), as described in Example VII.
[0056] FIG. 7C is a chemical structure ofN [[4-[[[[4,7,10-Tris(carboxymethyl)-
1,4,7,10-tetraazacyclododec- I -yl] acetyl] amino]methylJphenoxy] acetyl]-L-
glutaminyl-L-
5 tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide
(L124), as
described in Example VII.
[0057] FIG. 8A is a graphical representation of a sequence of reactions for
the
synthesis of intermediate 4-[[[9H Fluoren-9-ylmethoxy)carbonyl]amino]methyl]-3-
methoxybenzoic acid (E), as described in Example VIII.
10 [0058] FIG. 8B is a graphical representation of the synthesis ofN [4-
[[[[4,7,10-
Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-I -yl] acetyl] amino]methyl]-3-
methoxybenzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-
leucyl-L-
methioninamide, (L125), as described in Example VIII.
[0059] FIG. SC is a chemical structure ofN [4-[[[[4,7,10-Tris(carboxymethyl)-
1,4,7,10-tetraazacyclododec-1-ylJ acetyl] amino]methyl]-3-methoxybenzoylJ-L-
glutaminyl-L-
tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-Ieucyl-L-methioninamide,
(L125), as
described in Example VIII.
[0060] FIG. 9A is a graphical representation of a reaction for the synthesis
of 3-
[[[(9H Fluoren-9-ylmethoxy)carbonyl]amino]acetylJaminobenzoic acid, (B), as
described in
Example IX.
[0061 ] FIG. 9B is a graphical representation of a reaction for the synthesis
of 6-
[[[(9H Fluoren-9-ylmethoxy)carbonylJaminoJacetyl]aminonaphthoic acid (C), as
described in
Example IX.
[0062] FIG. 9C is a graphical representation of a reaction for the synthesis
of 4-
[[[[(9H Fluoren-9-ylmethoxy)carbonyl]amino]acetylJmethylamino]benzoic acid3
(D), as
described in Example IX.
[0063] FIG. 9D is a graphical representation of a reaction for the synthesis
of N [4-
[[[[ [4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-
y1] acetyl] amino] acetyl] amino]phenylacetyl]-L-glutaminyl-L-tryptophyl-L-
alanyl-L-valyl-
glycyl-L-histidyl-L-leucyl-L-methioninamide, (L146); N [3-[[[[[4,7,10-
Tris(carboxymethyl)-
1,4,7,10-tetraazacyclododec-1-ylJ acetyl] amino]acetyl]amino]benzoyl]-L-
glutaminyl-L-
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tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide
(L233); N [6-
[ [[ [ [4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl] acetyl]
amino] acetyl]
amino]naphthoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-
L-leucyl-L-
methioninamide, (L234), andN [4-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-
tetraazacyclododec-1-yl]acetyl]amino]acetyl] methylamino]benzoyl]-L-glutaminyl-
L-
tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide,
(L235), as
described in Example IX.
[0064] FIG. 10A is a graphical representation of a reaction for the synthesis
of 7-
[[Bis( I , l -dimethyl ethoxy)phosphinyl]methyl]-1,4,7,10-
tetraazacyclododecane-1,4,10-
triacetic acid 4,10-bis(I,l-dimethylethyl) ester H, as described in Example X.
[0065] FIG. lOB is a graphical representation of a reaction for the synthesis
ofN [4-
[[[[[4,10-Bis(carboxymethyl)-7-(dihydroxyphosphinyl)methyl-1,4,7,10-
tetraazacyclododec-
I -yl] acetyl] amino]acetyl] amino]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-
L-valyl-
glycyl-L-histidyl-L-leucil-L-methioninamide, (L237), as described in Example
X.
[0066] FIG.11A is a graphical representation of a reaction for the synthesis
of N,N
Dimethylglycyl-L-serinyl-[S-[(acetylamino)methyl]]-L-cysteinyl-glycyl-4-
aminobenzoyl-L-
glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-
methioninamide
(L238), as described in Example XI.
[0067] FIG. 11B is a graphical representation of a reaction for the synthesis
ofN,lV
Dimethylglycyl-L-serinyl-[S-[(acetylamino)methyl]]-L-cysteinyl-glycyl-
(313,513,7a,12a)-3-
amino-7,12-dihydroxy-24-oxocholan-24-yl-L-glutaminyl-L-tryptophyl-L-alanyl-L-
valyl-
glycyl-L-histidyl-L-leucyl-L-methioninamide, (L239), as described in Example
XI.
[0068] FIG. 12A is a graphical representation of a reaction for the synthesis
of 4-
[[[(9H Fluoren-9-ylmethoxy)carbonyl]amino]acetyl]amino-3-methoxybenzoic acid
(A), as
described in Example XII.
[0069) FIG. 12B is a graphical representation of a reaction for the synthesis
of 4-[[
[(91I Fluoren-9-ylmethoxy)carbonyl]amino]acetyl]amino-3-chlorobenzoic acid,
(D), as
described in Example XII.
[0070] FIG. 12C is a graphical representation of a reaction for the synthesis
of 4-
[[[(9H Fluoren-9-ylmethoxy)carbonyl]amino]acetyl]amino-3-methylbenzoic acid
(E), as
described in Example XII.
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[007I] FIG. 12D is a chemical structure ofN [4-[[[[4,7,10-Tris(carboxymethyl)-
1,4,7,10-tetraazacyclododec-1-y1] acetyl]glycyl]amino]-3-methoxybenzoyl]-L-
glutaminyl-L-
tryptophyl-I-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L240)
as
described in Example XII.
[0072] FIG. 12E is a chemical structure of compound N [4-[[[[4,7,10-
Tris(carboxymethyl)-1,4,7,lOtetraazacyclododec-1-yl] acetyl]glycyl]amino]3-
chlorobenzoyl]L-glutaminyl-L-tryptophyl-I-alanyl-L-valyl-glycyl-L-histidyl-L-
leucyl-L-
methioninamide, (L241) as described in Example XII.
[0073] FIG. 12F is a chemical structure ofN-[4-[[[[4,7,10-Tris(carboxymethyl)-
1,4,7,lOtetraazacyclododec-1-yl] acetyl]glycyl]amino]3-methylbenzoyl]L-
glutaminyl-L-
tryptophyl-I-alanyl-L-valyl-glycyl-L-histidyl-leucyl-L-methioninamide (L242),
as described
in Example XII.
[0074] FIG. 13A is a graphical representation of a reaction for the synthesis
of 4-
[N,N'-Bis[2-[(9 H fluoren-9-ylmethoxy)carbonyl]aminoethyl]amino]-4-oxobutanoic
acid,
(D), as described in Example XIII.
[0075] FIG. 13B is a graphical representation of a reaction for the synthesis
ofN [4-
[[4-[Bis [2-[[[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacycIododec-1-
yl]acetyl]amino]ethyl]amino-1,4-dioxobutyl]amino]benzoyl]-L-glutaminyl-L-
tryptophyl-L-
alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide, (L244), as
described in
Example XIII.
[0076] FIG. 13C is a chemical structure of compound L244, as described in
Example
XIII.
[0077] FIG. 14A and FIG. 14B are graphical representations of the binding and
competition curves described in Example XLIII.
[0078] FIG. 15A is a graphical representation of the results of radiotherapy
experiments described in Example LV.
[0079] FIG. 15B is a graphical representation of the results of other
radiotherapy
experiments described in Example LV.
[0080] FIG. 16 is a chemical structure ofN-[4-[[[[4,7,10-Tris(carboxymethyl)-
1,4,7,10 tetraazacyclododec-1-yl] acetyl]glycyl]amino]-L-Lysinyl-(3,6,9)-
trioxaundecane-
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1,1 I-dicarboxylic acid-3,7-dideoxy-3-aminocholic acid)-L-arginyl-L-glutaminyl-
L-
triptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L65).
[0081] FIG. 17 is a chemical structure ofN [2-S-[[[[(12a-Hydroxy-17a-(1-methyl-
3-
carboxypropyl)etiocholan-313-carbamoylmethoxyethoxyethoxyacetyl]-amino -6-
[4,7,10-
tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-
yl]acetyl]amino)acetyl]amino] hexanoyl-
L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-
methioninamide
(L66).
[0082] FIG.18A is a chemical structure ofN [4-[[[[[4,7,10-Tris(carboxymethyl)-
1,4,7,10-tetraazacyclododec-1-yl] acetyl] amino] acetyl]amino)benzoyl]-L-
glutaminyl-L-
tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L70).
[0083] FIG.18B is a chemical structure N [4-([[[[4,7,10-Tris(carboxymethyl)-
1,4,7,10-tetraazacyclododec-1-yl]-3-carboxypropionyl)amino]
acetyl]amino)benzoyl]-L-
glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-
methioninamide
(L114).
[0084] FIG.18C is a chemical structureN [4-[[4,7,10-Tris(carboxymethyl)-
1,4,7,10
tetraazacyclododec-1-yl)-2-hydroxy-3-propoxy]benzoyl)-L-glutaminyl-L-
tryptophyl-L
alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L144).
[0085) FIG.18D is a chemical structure N [(313,513, 7a, l2a)-3-[[[[[[4,7,10-
Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-
yl]acetyl)amino)ethoxyethoxy]acetyl)amino)-7,12-dihydroxycholan-24-yl]-L-
glutaminyl-L-
tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L69).
[0086] FIG. 18E is a chemical structure ofN [4-([[[(4,7,10-Tris(carboxymethyl)-
1,4,7,10-tetraazacyclododec-1-yl] acetyl] amino] acetyl) amino)phenylacetyl]-L-
glutaminyl-L-
tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide
(L146).
[0087] FIG. 19 dicloses chemical structures of intermediates which may be used
to
prepare compounds L64 and L70 as described in Example LVI.
[0088] FIG. 2O is a graphical representation of the preparation of L64 using
segment
coupling as described in Example LVI.
[0089) FIG. 21 is a graphical representation of the preparation of (1R)-1-(Bis
f 2-
[bis(carboxymethyl)amino]ethyl}amino)propane-3-carboxylic acid-1-carboxyl-
glycyl-4-
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aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-
Ieucyl-L-
methioninamide (L201).
[0090] FIG. 22A is a graphical representation of chemical structure of
chemical
intermediates used to prepare L202.
[0091 ] FIG. 22B is a graphical representation of the preparation of N-
[(3J3,513,12a)-3-
[[[[ [4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-
yl]acetyl]amino]acetyl]amino]-4-hydrazinobenzoyl-L-glutaminyl-L-tryptophyl-L-
alanyl-L-
valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L202).
[0092] FIG. 23A is a graphical representation of chemical structure of
chemical
intermediates used to prepare L203.
[0093] FIG. 23B is a graphical representation of the preparation of N-
[(3J3,513,12a)-3-
[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl] acetyl] amino]-
4-
aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-
leucyl-L-
methioninamide (L203).
[0094] FIG. 24 is a graphical representation of the preparation of N-
[(313,513,12a)-3-
[[ [4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl] acetyl]
amino]-4-
aminobenzoyl-glycyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-
histidyl-L-
leucyl-L-methioninamide (L204).
[0095] FIG. 25 is a graphical representation of the preparation of N-
[(313,513,12a)-3-
[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-4-
aminobenzoyl-glycyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-
histidyl-L-
leucyl-L-methioninamide (L205).
[0096] FIG. 26A is a graphical representation of chemical structures of
chemical
intermediates used to prepare L206.
[0097] FIG. 26B is a graphical representation of the preparation ofN-
[(313,513,12a)-3-
[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-
yl]acetylJamino]acetyl]amino]- [4'-Amino-2'-methyl biphenyl-4-carboxyl]-L-
glutaminyl-L-
tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide
(L206).
[0098] FIG. 27A is a graphical representation of chemical structures of
chemical
intermediates used to prepare L207.
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[0099] FIG. 27B is a graphical representation of the preparation of N-
[(313,513,12a)-3-
[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1- '
yl]acetyl]amino]acetyl]amino]- [3'-amino-biphenyl-3-carboxyl]-L-glutaminyl-L-
tryptophyl-
L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L207).
5 [00100] FIG. 28 is a graphical representation of the preparation of N-
[(313,513,12a)-3-
[[[ [[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-
yl]acetyl]amino]acetyl]amino]- [1,2-diaminoethyl-terephthalyl]-L-glutaminyl-L-
tryptophyl-
L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L208).
[00101] FIG. 29A is a graphical representation of chemical structures of
chemical
10 intermediates used to prepare L209.
[001021 FIG. 29B is a graphical representation of the preparation of L209.
[00103] FIG. 30A is a graphical representation of chemical structures of
chemical
intermediates used to prepare L210.
[00104] FIG. 30B is a chemical structure of L210.
15 [00105] FIG. 31 is a chemical structure ofN-[(313,S13,12a)-3-[[[4,7,10-
Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl] acetyl] amino]-glycyl-
glycyl-4-
aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-
leucyl-L-
methioninamide L211.
[00106] FIG. 32 is a chemical structure ofN-[(313,513,12a)-3-[[[4,7,10-
Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-
aminobenzoyl-L-glutamyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-
leucyl-L-
methioninamide L212.
[00107] FIG. 33 is a chemical structure ofN-[(3J3,S13,12a)-3-[[[4,7,10-
Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-
aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-
leucyl-L-
methionine carboxylate L213.
[001 O8] FIG. 34 is a chemical structure of N-[(3l3,513,12a)-3-[[[4,7,10-
Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl] acetyl]amino]-glycyl-4-
aminobenzoyl-D-phenyl al aryl-L-glutaminyl-L-tryptophyl-L-al anyl-L-valyl-
glycyl-L-
histidyl-L-leucyl-L-methioninamide L214.
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[00109] FIG. 35 is a chemical structure ofN-[(3J3,513,12a)-3-[[[4,7,10-
Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec- I -yl] acetyl] amino]-glycyl-
4-
aminobenzoyl-L-glutaminyl-L-arginyl-L-leucyl-glycyl-L-asparginyl-L-glutaminyl-
L-
tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide L215.
[00110] FIG. 36 is a chemical structure ofN-[(313,513,12a)-3-[[[4,7,10-
Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl] acetyl] amino]-glycyl-4-
aminobenzoyl-L-glutaminyl-arginyl-L-tyrosinyl-glycyl-L-asparginyl-L-glutaminyl-
L-
tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide L216.
[00111] FIG. 37 is a chemical structure ofN-[(313,513,12a)-3-[[[4,7,10-
Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-
aminobenzoyl-L-glutaminyl-L-lysyl-L-tyrosinyl-glycyl-L-glutaminyl-L-tryptophyl-
L-alanyl-
L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide L217.
[00112] FIG. 38 is a chemical structure of L218.
[00113] FIG. 39 is a chemical structure ofN-[(3J3,513,12a)-3-[[[4,7,10-
Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-
aminobenzoyl-D-phenylalanyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-
L-
histidyl-L-leucyl-aminopentyl, L219.
[00114] FIG. 40 is a chemical structure of N-[(3J3,5l3,12a)-3-[[[4,7,10-
Tris(carboxym ethyl)-1,4, 7,10-tetraazacyclododec-1-yl] acetyl] amino]-glycyl-
4-
aminobenzoyl-L-glutaminyl-L-tryptophyl-L-serinyl-L-valyl-D-alanyl-L-histidyl-L-
leucyl-L-
methioninamide, L220.
[00115] FIG. 41 is a chemical structure ofN-[(313,SJ3,12a)-3-[[[4,7,10-
Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl] acetyl]amino]-glycyl-4-
aminobenzoyl-D-phenyl al anyl-L-glutaminyl-L-tryptophyl-L-al anyl-L-val yl-
glycyl-L-
histidyl-L-leucyl-L-leucinamide, L221.
[00116] FIG. 42 is a chemical structure of N-[(313,5l3,12a)-3-[[[4,7,10-
Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl] acetyl]amino]-glycyl-4-
aminobenzoyl-D-tyrosinyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-betaalanyl-
L-
histidyl-L-phenylalanyl-L-norleucinamide, L222.
[00117] FIG. 43 is a chemical structure ofN-[(3J3,513,12a)-3-[[[4,7,10-
Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl] acetyl] amino]-glycyl-4-
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aminobenzoyl-L-phenylalanyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-
betaalanyl-L-
histidyl-L-phenylalanyl-L-norleucinamide, L223.
[00118] FIG. 44 is a chemical structure ofN-[(313,513,12a)-3-[[[4,7,I0-
Tris(carboxymethyl)-I ,4,7,10-tetraazacyclododec-1-yl] acetyl] amino]-glycyl-4-
aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-glycyl-L-histidyl-L-
phenylalanyl-L-
leucinamide, L224.
[00119] FIG. 45 is a chemical structure ofN-[(313,513,12a)-3-[[[4,7,10-
Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl] acetyl] amino]-glycyl-4-
aminob enzoyl-L-1 eucyl-L-tryptophyl-L-al anyl-L-valinyl-glycyl-L-s erinyl-L-
phenyl al anyl-L-
methioninamide, L225.
[00120] FIG. 46 is a chemical structure ofN-[(313,513,12a)-3-[[[4,7,10-
Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl] acetyl] amino]-glycyl-4-
aminobenzoyl-L-histidyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-
leucyl-L-
methioninamide, L226.
[00121] FIG. 47 is a chemical structure ofN-[(313,513,12a)-3-[[[4,7,10-
Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl] acetyl] amino]-glycyl-4-
aminobenzoyl-L-leucyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-serinyl-L-
phenylalanyl-L-
methioninamide L227.
[00122] FIG. 48 is a chemical structure ofN-[(313,513,12a)-3-[[[4,7,10-
Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-
aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-
phenylalanyl-
L-methioninamide, L228.
[00123] FIG. 49A is a graphical representation of a reaction for the synthesis
of (313,513
7a,12a)-3-(9H-Fluoren-9-ylmethoxy)amino-7,12-dihydroxycholan-24-oic acid (B)
as
described in Example LVII.
[00124] FIG. 49B is a graphical representation of a reaction for the synthesis
of N-[313,513
7a,12a)-3-[[[2-[2-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-
yl]acetyl]amino]ethoxy]ethoxy]acetyl]amino]-7,12-dihydroxy-24-oxocholan-24-yl]-
L-
glutaminyl-L-tryptophyl-L-al anyl-L-valyl-glycyl-L-hi stidyl-L-1 eucyl-L-methi
oninamide,
(L69), as described in Example LVII.
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[00125] FIG. 50 is a graphical representation of a reaction for the synthesis
of N-[4-[2-
Hydroxy-3-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-
yl]propoxy]benzoyl]-
L-glutaminyl-L-tryptophyI-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-
methioninamide
(L144), as described in Example LVIII.
[00126 FTG. 51 is a chemical structure of L300.
[00127] FIG. 52 is a TOCSY spectrum of Lu-L70 in DMSO-d6 at 25°C.
[0012&] FIG. 53 is a COSY spectrum of Lu-L70 in DMSO-d6 at 25°C.
[00129] FIG. 54 is a NOESY spectrum of Lu-L70 in DMSO-d6 at 25°C.
[00130] FIG. 55 is a gHSQC spectrum of Lu-L70 in DMSO-d6 at 25°C.
[00131] FIG. 56 is a gHMBC spectrum of Lu-L70 in DMSO-d6 at 25 °C.
[00132] FIG. 57 is a gHSQCTOCSY spectrum of Lu-L?0 in DMSO-d6 at 25°C.
[00133] FIG. 58 is a Regular 1H-NMR (bottom) and selective homo-decoupling of
the
water peak at 3.5 ppm of Lu-L70 in DMSO-d6 at 15 °C.
[00134] FIG. 59 is a TOCSY Spectrum of ~~SLu-D03A-monoamide-Aoc-QWAVGHLM-
NH2 in DMSO-d& at 25 °C.
[00135] FIG. 60 is a chemical structure of L70.
[00136] FIG. 61 is a chemical structure of ~~SLu-D03A-monoamide-Aoc-QWAVGHLM-
NH2.
[00137] FIG. 62 is a chemical structure of ~~SLu-L70 with a bound water
molecule.
~ [00138] FIG. 63 is a chemical structure of L301.
Abbreviations Used In the Application
Aoc- ~-aminooctanoic acid
A a3- 3-amino ro ionic acid
Abu4- 4-aminobutanoic acid
Adca3- (313,513 7a,,12a,)-3-amino-7,12-dihydroxycholan-24-oic
acid
or 3-Amino-3-deox cholic acid
Ahl2ca- 3J3,5J3,12a -3-amino-12-h drox cholan-24-oic
acid
Akca- (3f3,513,7a,12a)-3-amino-12-oxacholan-24-oic
acid
Cha- L-C clohex lalanine
Nall- ~ L-1-Naphthylalanine
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Bi - L-Bi hen lalanine
Mo3abz4- 3-Methoxy-4-aminobenzoic acid or 4-aminomethyl-3-
methox benzoic acid
B a4- 4-benzo 1 hen lalanine
C13 abz4- 3-Chloro-4-aminobenzoic acid
M3abz4- 3-meth 1-4-aminobenzoic acid
Ho3abz4- 3-h drox -4-aminobenzoic acid
Hybz4- 4-h drazinobenzoic acid
Nmabz4- 4-meth laminobenzoic acid
Mo3amb4- 3-methox -4-aminobenzoic acid
Amb4- 4-aminomethylbenzoic acid
Aeb4- 4-(2-aminoethox benzoic acid
Dae- 1,2-diaminoeth I
T a- Tere hthalic acid
A4m~bi hc4- 4'-Amino-2'-meth 1 bi hen 1-4-carbox lic
acid
A3bi hc3- 3-amino-3'-bi hen lcarbox lic acid
Amc4- trans-4-aminometh lc clohexane carbox Iic
acid
Ae a4- N-4-aminoethyl-N-1- i erazine-acetic acid
In - Isoni ecotic acid
Pial- N-1- i erazineacetic acid
Ckb - 4- 3-Carbox eth 1-2-keto-1-benzimidazo
1 - i eridine
Abz3 3-Aminobenzoic acid
Abz4 4-Aminobenzoic acid
J 8-amino-3,6-dioxaoctanoic acid
AvaS 5-Aminovaleric acid
f
(D -Phe
D -T
Ala2 (also Bala) Beta-alanine
DETAILED DESCRIPTION OF THE INVENTION
[00139] In the following description, various aspects of the present invention
will be
further elaborated. For purposes of explanation, specific configurations and
details are set
forth in order to provide a thorough understanding of the present invention.
However, it will
also be apparent to one skilled in the art that the present invention may be
practiced without
the specific details. Furthermore, well known features may be omitted or
simplified in order
not to obscure the present invention.
[00140] In an embodiment of the present invention, there are provided new and
improved
compounds for use in diagnostic imaging or radiotherapy. The compounds include
an optical
label or a chemical moiety capable of complexing a medically useful metal ion
or
CA 02549318 2006-06-08
WO 2005/067983 PCT/US2004/022115
radionuclide (metal chelator) attached to a GRP receptor targeting peptide by
a linker or
spacer group.
[00141 ] In general, compounds of the present invention may have the formula:
M-N-O-P-G
5 wherein M is the metal chelator (in the form complexed with a metal
radionuclide or not), or
an optical label, N-O-P is the linker, and G is the GRP receptor targeting
peptide. Each of the
metal chelator, optical label, linker, and GRP receptor targeting peptide is
described in the
discussion that follow.
[00I42] In another embodiment of the present invention, there is provided a
new and
10 improved linker or spacer group which is capable of linking an optical
label or a metal
chelator to a GRP receptor targeting peptide. In general, linkers of the
present invention may
have the formula:
N-O-P
wherein each of N, O and P are defined throughout the specification.
15 [00143] Compounds meeting the criteria defined herein were discovered to
have improved
pharmacokinetic properties compared to other GRP receptor targeting peptide
conjugates
known in the art. For example, compounds containing the linkers of the present
invention
were retained in the bloodstream longer, and thus had a longer half life than
prior known
compounds. The longer half life was medically beneficial because it permitted
better tumor
20 targeting which is useful for diagnostic imaging, and especially for
therapeutic uses, where
the cancerous cells and tumors receive greater amounts of the radiolabeled
peptides.
Additionally, compounds of the present invention had improved tissue receptor
specificity
compared to prior art compounds.
1 A. Metal Chelator
[00144] The term "metal chelator" refers to a molecule that forms a complex
with a metal
atom, wherein said complex is stable under physiological conditions. That is,
the metal will
remain complexed to the chelator backbone in vivo. More particularly, a metal
chelator is a
molecule that complexes to a radionuclide metal to form a metal complex that
is stable under
physiological conditions and which also has at least one reactive functional
group for
conjugation with the linker N-O-P. The metal chelator M may be any of the
metal chelators
known in the art for complexing a medically useful metal ion or radionuclide.
The metal
CA 02549318 2006-06-08
WO 2005/067983 PCT/US2004/022115
21
chelator may or may not be complexed with a metal radionuclide. Furthermore,
the metal
chelator can include an optional spacer such as, for example, a single amino
acid (e.g., Gly)
which does not complex with the metal, but which creates a physical separation
between the
metal chelator and the linker.
[00145] The metal chelators of the invention may include, for example, linear,
macrocyclic, terpyridine, and N3S, N2Sz, or N4 chelators (see also, U.S.
5,367,080, U.S.
5,364,613, U.S. 5,021,556, U.S. 5,075,099, U.S. 5,886,142, the disclosures
ofwhich are
incorporated by reference in their entirety), and other chelators known in the
art including,
but not limited to, HYNIC, DTPA, EDTA, DOTA, TETA, and bisamino bisthiol (BAT)
chelators (see also U.S. 5,720,934). For example, Nd chelators are described
in U.S. Patent
Nos. 6,143,274; 6,093,382; 5,608,110; 5,665,329; 5,656,254; and 5,688,487, the
disclosures
of which are incorporated by reference in their entirety. Certain N3S
chelators are described
in PCT/CA94100395, PCT/CA94/00479, PCTlCA95/00249 and in U.S. Patent Nos.
5,662,885; 5,976,495; and 5,780,006, the disclosures of which are incorporated
by reference
in their entirety. The chelator may also include derivatives of the chelating
ligand mercapto-
acetyl-glycyl-glycyl-glycine (MAG3), which contains an N3S, and N2S2 systems
such as
MAMA (monoamidemonoaminedithiols), DADS (NZS diaminedithiols), CODADS and the
like. These ligand systems and a variety of others are described in Liu and
Edwards, Chem
Rev. 1999, 99, 2235-2268 and references therein, the disclosures ofwhich are
incorporated
by reference in their entirety.
[00146] The metal chelator may also include complexes containing ligand atoms
that are
not donated to the metal in a tetradentate array. These include the boronic
acid adducts of
technetium and rhenium dioximes, such as those described in U.S. Patent Nos.
5,183,653;
5,387,409; and 5,118,797, the disclosures of which are incorporated by
reference in their
entirety.
[00147] Examples ofpreferred chelators include, but are not limited to,
diethylenetriamine
pentaacetic acid (DTPA), 1,4,7,10-tetraazacyclotetradecane-1,4,7,10-
tetraacetic acid
(DOTA), 1-substituted 1,4,7,-tricarboxymethyl 1,4,7,10-tetraazacyclododecane
triacetic acid
(D03A), ethylenediaminetetraacetic acid (EDTA), 4-carbonylmethyl-10-
phosponomethyl-
1,4,7,10-Tetraazacyclododecane-1,7-diacetic acid (Cm4pml0d2a); and 1,4,8,11-
tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA). Additional
chelating ligands are
ethylenebis-(2-hydroxy-phenylglycine) (EHPG), and derivatives thereof,
including
5-Cl-EHPG, 5-Br-EHPG, 5-Me-EHPG, 5-t-Bu-EHPG, and 5-sec-Bu-EHPG;
CA 02549318 2006-06-08
WO 2005/067983 PCT/US2004/022115
22
benzodiethylenetriamine pentaacetic acid (benzo-DTPA) and derivatives thereof,
including
dibenzo-DTPA, phenyl-DTPA, diphenyl-DTPA, benzyl-DTPA, and dibenzyl-DTPA; bis-
2
(hydroxybenzyl)-ethylene-diaminediacetic acid (HBED) and derivatives thereof;
the class of
macrocyclic compounds which contain at least 3 carbon atoms, more preferably
at least 6,
and at least two heteroatoms (O and/or N), which macrocyclic compounds can
consist of one
ring, or two or three rings joined together at the hetero ring elements, e.g.,
benzo-DOTA,
dibenzo-DOTA, and benzo-NOTA, where NOTA is 1,4,7-triazacyclononane
N,N',N"-triacetic acid, benzo-TETA, benzo-DOTMA, where DOTMA is
1,4,7,10-tetraazacyclotetradecane-1,4,7, 10-tetra(methyl tetraacetic acid),
and
benzo-TETMA, where TETMA is 1,4,8,11- tetraazacyclotetradecane-1,4,8,11-
(methyl
tetraacetic acid); derivatives of 1,3-propylenediaminetetraacetic acid (PDTA)
and
triethylenetetraaminehexaacetic acid (TTHA); derivatives of
1,5,10-N,N',N"-tris(2,3-dihydroxybenzoyl)-tricatecholate (LICAM) and
1,3,5-N,N',N"-tris(2,3-dihydroxybenzoyl) aminomethylbenzene (MECAM). Examples
of
representative chelators and chelating groups contemplated by the present
invention are
described in WO 98/18496, WO 86/06605, WO 91/03200, WO 95/28179, WO 96/23526,
WO 97/36619, PCT/US98/01473, PCTlLJS98/20182, and U.S. 4,899,755, U.S.
5,474,756,
U.S. 5,846,519 and U.S. 6,143,274, each of which is hereby incorporated by
reference in its
entirety.
[00148] Particularly preferred metal chelators include those of Formula l, 2
and 3 (for
~~lIn and radioactive lanthanides, such as, for example ~~~Lu, 901', ~s3Sm,
and ~66Ho) and
those of Formula 4, 5 and 6 (for radioactive 99"'Tc, ~ $6Re, and 188Re) set
forth below. These
and other metal chelating groups are described in U.S. Patent Nos. 6,093,382
and 5,608,110,
which are incorporated by reference in their entirety. Additionally, the
chelating group of
formula 3 is described in, for example, U.S. Patent No. 6,143,274; the
chelating group of
formula 5 is described in, for example, U.S. Patent Nos. 5,627,286 and
6,093,382, and the
chelating group of formula 6 is described in, for example, U.S. Patent Nos.
5,662,885;
5,780,006; and 5,976,495, all of which are incorporated by reference. Specific
metal
chelators of formula 6 include N,N-dimethylGly-Ser-Cys; N,N-dimethylGly-Thr-
Cys ; N,N-
diethylGly-Ser-Cys; N,N-dibenzylGly-Ser-Cys; and other variations thereof. For
example,
spacers which do not actually complex with the metal radionuclide such as an
extra single
amino acid Gly, may be attached to these metal chelators (e.g., N,N-
dimethylGly-Ser-Cys-
Gly; N,N-dimethylGly-Thr-Cys-Gly; N,N-diethylGly-Ser-Cys-Gly; N,N-dibenzylGIy-
Ser-
CA 02549318 2006-06-08
WO 2005/067983 PCT/US2004/022115
23
Cys-Gly). Other useful metal chelators such as all of those disclosed in U.S.
Pat. No.
6,334,996, also incorporated by reference (e.g., Dimethylgly-L-t-Butylgly-L-
Cys-Gly;
Dimethylgly-D-t-Butylgly-L-Cys-Gly; Dimethylgly-L-t-Butylgly-L-Cys, etc.)
[00149] Furthermore, sulfur protecting groups such as Acm (acetamidornethyl),
trityl or
other known alkyl, aryl, acyl, alkanoyl, aryloyl, mercaptoacyl and organothiol
groups may be
attached to the cysteine amino acid of these metal chelators.
[00150] Additionally, other useful metal chelators include:
R R
H OOC---~ ~'~ ~--- COOH
N N
C
,N N
HOOC~ ~ COOH
~R CO
(1)
R R
HOOC----~ n ~--COOH
N N
C
N N
HOOC~ U COOH
~R NH
(2)
HOOC--~ n ~--COOH
N N
~---COOH
(3)
CA 02549318 2006-06-08
WO 2005/067983 PCT/US2004/022115
24
O
HN ~ ~-O
n
NH HN NH HN
~N N~ ~ ~N N~
HO OH O HO OH
(4a) (4b)
YiJ ~n
X~ X
NH HN
NH HN
iJ ~~ /
N Y ~~ I I
HO OH HO OH
(5a) (5b)
OH
O O
O N N~ °
~\
_ a
/N S/
NHCOCH3
(6)
CA 02549318 2006-06-08
WO 2005/067983 PCT/US2004/022115
O
O NH HN
HS
(7)
[00151] In the above Formulas I and 2, R is alkyl, preferably methyl. In the
above
5 Formulas Sa and Sb, X is either CH2 or O; Y is C1-Clo branched or unbranched
alkyl; aryl,
aryloxy, arylamino, arylaminoacyl; arylalkyl - where the alkyl group or groups
attached to
the aryl group are C1-Clo branched or unbranched alkyl groups, C1-Clo branched
or
unbranched hydroxy or polyhydroxyalkyl groups or polyalkoxyalkyl or
polyhydroxy-
polyalkoxyalkyl groups; J is optional, but if present is C(=O)-, OC(=O)-, SOZ-
, NC(=O)-,
10 NC(=S)-, N(Y), NC(=NCH3)-, NC(=NH)-, N=N-, homopolyamides or
heteropolyamines
derived from synthetic or naturally occurring amino acids; all where n is 1-
100. Other
variants of these structures are described, for example, in U.S. Patent No.
6,093,382. In
Formula 6, the group S-NHCOCH3 may be replaced with SH or S-Z wherein Z is any
of the
known sulfur protecting groups such as those described above. Formula 7
illustrates one
15 embodiment of t-butyl compounds useful as a metal chelator. The disclosures
of each of the
foregoing patents, applications and references are incorporated by reference
in their entirety.
[00152] In a preferred embodiment, the metal chelator includes cyclic or
acyclic
polyaminocarboxylic acids such as DOTA (1,4,7,10-tetraazacyclododecane -
1,4,7,10-
tetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), DTPA-
bismethylamide, DTPA-
20 bismorpholineamide, Cm4pm10d2a (1,4-carbonylmethyl-10-phosponomethyl-
1,4,7,10-
Tetraazacyclododecane-1,7-diacetic acid), D03A N [[4,7,10-Tris(carboxyrnethyl)-
1,4,7,10-
tetraazacyclododec-1-yl]acetyl, HP-DO3A, D03A-monoamide and derivatives
thereof.
[00153] Preferred metal radionuclides for scintigraphy or radiotherapy include
9~"'Tc, SICr,
67Ga 68~a 47SC SlCr 167Tm 141Ce 111In 16s~ 175 140La 90Y 88Y 153Sm 166HO 165D
> > > > > > > > > > > > > > y~
25 166D 62~u 64Cu 67~u 97Ru 103Ru 186Re 188Re 203Pb 211$i 212$i 213$i 214Bi
105 109Pd
y> > > > > > > > > > > > > > >
l lTnSn, 149Pm, 161Tb, 177Lu, l9sAu and 199Au and oxides or nitrides thereof.
The choice of
metal will be determined based on the desired therapeutic or diagnostic
application. For
CA 02549318 2006-06-08
WO 2005/067983 PCT/US2004/022115
26
example, for diagnostic purposes (e.g., to diagnose and monitor therapeutic
progress in
primary tumors and metastases), the preferred radionuclides include 64Cu,
6~Ga, 68Ga, 99mTc,
and "'In, with 99mTc and "'In being especially preferred. For therapeutic
purposes (e.g., to
provide radiotherapy for primary tumors and metastasis related to cancers of
the prostate,
breast, lung, etc.), the preferred radionuclides include 64Cu, 9oY~ ~os~~ mln,
I'~mSn, ~49Pm,
~s3sm~ ~6~.Lb~ is6Dy~ ~66Ho~ ms~,b~ I~~Lu, ~s6nssRe, and ~99Au, with 3?~Lu and
9°Y being
particularly preferred. 99mTc is particularly useful and is a preferred for
diagnostic
radionuclide because of its low cost, availability, imaging properties, and
high specific
activity. The nuclear and radioactive properties of 99"'Tc make this isotope
an ideal
scintigraphic imaging agent. This isotope has a single photon energy of 140
keV and a
radioactive half life of about 6 hours, and is readily available from a 99Mo
99mTc generator.
Fox example, the 99"'Tc labeled peptide can be used to diagnose and monitor
therapeutic
progress in primary tumors and metastases. Peptides labeled with ~~~Lu,
9°Y or other
therapeutic radionuclides can be used to provide radiotherapy for primary
tumors and
metastasis related to cancers of the prostate, breast, lung, etc.
1B. Optical Labels
[00154] In an exemplary embodiment, the compounds of the invention may be
conjugated
with photolabels, such as optical dyes, including organic chromophores or
fluorophores,
having extensive delocalized ring systems and having absorption or emission
maxima in the
range of 400-1540 nm. The compounds of the invention may alternatively be
derivatized
with a bioluminescent molecule. The preferred range of absorption maxima for
photolabels
is between 600 and 1000 nm to minimize interference with the signal from
hemoglobin.
Preferably, photoabsorption labels have large molar absorptivities, e.g. > !Os
crri 1M-!, while
fluorescent optical dyes will have high quantum yields. Examples of optical
dyes include,
but are not limited to those described in WO 98/18497, WO 98/18496, WO
98/18495, WO
98118498, WO 98/53857, WO 96!17628, WO 97/18841, WO 96/23524, WO 98/47538, and
references cited therein. For example, the photolabels may be covalently
linked directly to
compounds of the invention, such as, for example, compounds comprised of GRP
receptor
targeting peptides and linkers of the invention. Several dyes that absorb and
emit light in the
visible and near-infrared region of electromagnetic spectrum are currently
being used for
various biomedical applications due to their biocompatibility, high molar
absorptivity, and/or
high fluorescence quantum yields. The high sensitivity of the optical modality
in conjunction
with dyes as contrast agents parallels that of nuclear medicine, and permits
visualization of
CA 02549318 2006-06-08
WO 2005/067983 PCT/US2004/022115
27
organs and tissues without the undesirable effect of ionizing radiation.
Cyanine dyes with
intense absorption and emission in the near-infrared (NIR) region are
particularly useful
because biological tissues are optically transparent in this region. For
example, indocyanine
green, which absorbs and emits in the NIR region has been used for monitoring
cardiac
output, hepatic functions, and liver blood flow and its functionalized
derivatives have been
used to conjugate biomolecules for diagnostic purposes (R. B. Mujumdar, L. A.
Ernst, S. R.
Mujumdar, et al., Cyanine dye labeling reagents: Sulfoindocyanine succinimidyl
esters.
Bioconjugate Chemistry, 1993, 4(2), 105-11 l; Linda G. Lee and Sam L. Woo. "N-
Heteroaromatic ion and iminium ion substituted cyanine dyes for use as
fluorescent labels",
U.S. Pat. No. 5,453,505; Eric Hohenschuh, et al. "Light imaging contrast
agents", WO
98/48846; Jonathan Turner, et al. "Optical diagnostic agents for the diagnosis
of
neurodegenerative diseases by means of near infra-red radiation", WO 98/22146;
Kai Licha,
et al. "In-vivo diagnostic process by near infrared radiation", WO 96/17628;
Robert A. Snow,
et al., Compounds, WO 98/48838. Various imaging techniques and reagents are
described in
U.S. Patents 6,663,847, 6,656,451, 6,641,798, 6,485,704, 6,423,547, 6,395,257,
6,280,703,
6,277,841, 6,264,920, 6,264,919, 6,228,344, 6,217,848, 6,190,641, 6,183,726,
6,180,087,
6,180,086, 6,180,085, 6,013,243, and published U.S. Patent Applications
2003185756,
20031656432, 2003158127, 2003152577, 2003143159, 2003105300, 2003105299,
2003072763, 2003036538, 2003031627, 2003017164, 2002169107, 2002164287, and
2002156117. All of the above references are incorporated by reference in their
entirety.
2A. Linkers Containing At Least One Non-alpha Amino Acid
[00155] In one embodiment of the invention, the linker N-O-P contains at least
one non-
alpha amino acid. Thus, in this embodiment of the linker N-O-P,
N is 0 (where 0 means it is absent), an alpha or non-alpha amino acid
or other linking group;
O is an alpha or non-alpha amino acid; and
P is 0, an alpha or non-alpha amino acid or other linking group,
wherein at least one of N, O or P is a non-alpha amino acid.
Thus, in one example, N = Gly, O = a non-alpha amino acid, and P= 0.
[00156] Alpha amino acids are well known in the art, and include naturally
occurring and
synthetic amino acids.
CA 02549318 2006-06-08
WO 2005/067983 PCT/US2004/022115
28
[00157 Non-alpha amino acids are also known in the art and include those which
are
naturally occurnng or synthetic. Preferred non-alpha amino acids include:
8-amino-3,6-dioxaoctanoic acid;
N-4-aminoethyI-N-1-acetic acid; and
polyethylene glycol derivatives having the formula NHZ-(CHzCHzO)n-
CHZCOzH or NHZ-(CHZCHZO)n-CH2CHZCOZH where n = 2 to
100.
[00158) Examples of compounds having the formula M-N-O-P-G which contain
linkers
with at least one non-alpha amino acid are listed in Table 1. These compounds
may be
prepared using the methods disclosed herin, particularly in the Examples, as
well as by
similar methods known to one skilled in the art.
TABLE 1
Table
1
-
Compounds
Containing
Linkers
With
At
Least
One
Non-alpha
Amino
Acid
Compo HPLC HPLC
and method'RTZ MS3 IC505M N O P G*
Ll 10-40%B5.431616.65 N,N- Lys 8-amino-3,6-none BBN(7-14)
dimethylglycine-Ser-~ dioxaoctanoic
C s Acm acid
-Gl
L2 10-40%B5.471644.73 N,N- Arg 8-amino-3,6-none BBN(7-14)
dimethylglycine-Ser- dioxaoctanoic
C s(Acm acid
-GI
L3 10-40%B5.971604.6>50 N,N- Asp 8-amino-3,6-none BBN(7-14)
dimethylglycine-Ser- dioxaoctanoic
C s Acm acid
-GI
LA 10-40%B5.921575.54 N,N- Ser 8-amino-3,6-none BBN(7-14)
dimethylglycine-Ser- dioxaoctanoic
C s(Acm acid
-GI
L5 10-40%B5.941545.59 N,N- Gly 8-amino-3,6-none BBN(7-14)
dimethylglycine-Ser- dioxaoctanoic
C s Acm acid
-Gl
L6 10-30%B7.821639(M>50 N,N- Glu 8-amino-3,6-none BBN(7-14)
+Na) dimethylglycine-Ser- dioxaoctanoic
C s Acm acid
-GI
L7 10-30%B8.471581 7 N,N- Dala 8-amino-3,6-none BBN(7-14)
(M+Na) dimethylglycine-Ser- dioxaoctanoic
C s Acm acid
-Gl
L8 10-30%B6.721639 4 N,N- 8-amino-3,6-Lys none BBN(7-14)
(M+Na) dimethylglycine-Ser-dioxaoctanoic
C s(Acm acid
-GI
L9 10-30%B7.28823.36 N,N- 8-amino-3,6-Arg none BBN(7-14)
~ (M+2/2) dimethylglycine-Ser-dioxaoctanoic
C s Acm acid
-GI
L10 10-30%B7.941625.6>50 N,N- 8-amino-3,6-Asp none BBN(7-14)
(M+Na) dimethylglycine-Ser-dioxaoctanoic
Cys(Acm acid
-Gly
CA 02549318 2006-06-08
WO 2005/067983 PCT/US2004/022115
29
7('able
1
-
Compounds
Containing
Linkers
With
At
Least
One
Non-alpha
Amino
Acid
CompoHPLC HPLC
and method'RTZ MS3 IC505M N O P G*
L1 10-30%B7.591575.636 N,N- 8-amino-3,6-Ser none BBN(7-14)
I
dimethylglycine-Ser-dioxaoctanoic
C s Acm -GI acid
L12 10-30%B7.651567.5>50 N,N- 8-amino-3,6-Gly none BBN(7-14)
(M+Na) dimethylglycine-Ser-dioxaoctanoic
C s(Acm -GI acid
L13 10-30%B7.861617.7>50 N,N- 8-amino-3,6-Glu none BBN(7-14)
dimethylglycine-Ser-dioxaoctanoic
Cys(Acm -Gly acid
L14 10-30%B7.9 1581.711 N,N- 8-amino-3,6-Dala none BBN(7-14)
(M+Na) dimethylglycine-Ser-dioxaoctanoic
C s Acm -GI acid
L15 10-30%B7.841656.811.5N,N- 8-amino-3,6-8-amino-3,6-none BBN(7-14)
(M+Na) dimethylglycine-Ser-dioxaoctanoicdioxaoctanoic
C s Acm -GI acid acid
L16 10-30%B6.651597.417 N,N- 8-amino-3,6-2,3- none BBN(7-14)
(M+Na) dimethylglycine-Ser-dioxaoctanoicdiaminopropio
C s Acm -GI acid nic acid
L17 10-30%B7.6 1488.68 N,N- none 8-amino-3,6-none BBN(7-14)
dimethylglycine-Ser- dioxaoctanoic
C s Acm -GI acid
L18 10-30%B7.031574.67.8 N,N- 2,3- 8-amino-3,6-none BBN(7-14)
dimethylglycine-Ser-diaminopropionidioxaoctanoic
C s Acm -GI c acid acid
L19 10-35%B5.131603.6>50 N,N- Asp 8-amino-3,6-Gly BBN(7-14)
dimethylglycine-Ser- dioxaoctanoic
C s Acm -GI acid
L20 10-35%B5.191603.637 N,N- 8-amino-3,6-Asp Gly BBN(7-14)
dimethylglycine-Ser-dioxaoctanoic
C s(Acm -Gl acid
L21 10-35%B5.041575.746 N,N- 8-amino-3,6-Ser Gly BBN(7-14)
dimethylglycine-Ser-dioxaoctanoic
C s Acm -Gl acid
L22 10-35%B4.371644.736 N,N- 8-amino-3,6-Arg Gly BBN(7-14)
dimethylglycine-Ser-dioxaoctanoic
C s(Acm -Gl acid
L23 10-35%B532 1633.7>50 N,N- 8-amino-3,6-8-amino-3,6-Gly BBN(7-14)
dimethylglycine-Ser-dioxaoctanoicdioxaoctanoic
C s(Acm -GI acid acid
L24 IO-35%B4.181574.638 N,N- 8-amino-3,6-2,3- Gly BBN(7-14)
dimethylglycine-Ser-dioxaoctanoicdiaminopropio
Cys(Acm -Gly acid nic acid
L25 10-35%B4.241616.626 N,N- 8-amino-3,6-Lys Gly BBN(7-14)
dimethylglycine-Ser-dioxaoctanoic
C s Acm -Gl acid
L26 10-35%B4.451574.630 N,N- 2,3- 8-amino-3,6-Gly BBN(7-14)
dimethylglycine-Ser-diaminopropionidioxaoctanoic
C s(Acm -Gl c acid acid
L27 10-35%B4.381627.3>50 N,N- N-4-aminoethyl-Asp none BBN(7-14)
dimethylglycine-Ser-N-1-
Cys(Acm)-Gly piperazineacetic
acid
CA 02549318 2006-06-08
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Table
1
-
Compounds
Containing
Linkers
With
At
Least
One
Non-alpha
Amino
Acid
CompoHPLC HPLC
and method'RTZ MS3 IC505M N O P G*
L28 10-35%B4.1 1600.325 N,N- N-4-aminoethyl-Ser none BBN(7-14)
d imethylglycine-Ser-N-1-
Cys(Acm)-Glypiperazineacetic
acid
L29 10-35%B3.711669.436 N,N- N-4-aminoethyl-Arg none BBN(7-14)
d imethylglycine-Ser-N-1-
Cys(Acm)-Glypiperazineacetic
acid
L30 10-35%B4.571657.236 N,N- N-4-aminoethyl-8-amino-3,6-none BBN(7-
14)
dimethylglycine-Ser-N-1- dioxaoctanoic
Cys(Acm)-Glypiperazineaceticacid
acid
L31 10-35%B3.691598.3>50 N,N- N-4-aminoethyl-2,3- none BBN(7-14)
dimethylglycine-Sex-N-1- diaminopropio
Cys(Acm)-Glypiperazineaceticnic acid
acid
L32 10-35%B3.511640.334 N,N- N-4-aminoethyl-Lys none BBN(7-14)
dimethylglycine-Ser-N-1-
Cys(Acm)-Glypiperazineacetic
acid
L33 10-35%B4.291584.5>50 N,N- N-1- Asp none BBN(7-14)
dimethylglycine-Ser-piperazineacetic
Cys(Acm)-Glyacid
L34 10-35%B4.071578.738 N,N- N-1- Ser none BBN(7-14)
(M+Na) dimethylglycine-Ser-piperazineacetic
C s(Acm -Gl acid
L35 10-35%B3.651625.626 N,N- N-1- Arg none BBN(7-14)
dimethylglycine-Ser-piperazineacetic
C s(Acm -Gl acid
L36 10-35%B4.431636.67 N,N- N-1- 8-amino-3,6-none BBN(7-14)
dimethylglycine-Ser-piperazineaceticdioxaoctanoic
Cys(Acm -Glyacid acid
L37 10-35%B3.661555.723 N,N- N-I- 2,3- none BBN(7-14)
dimethylglycine-Ser-piperazineaceticdiaminopropio
C s(Acm-Gl acid nic acid
L38 10-35%B3.441619.67 N,N- N-1- Lys none BBN(7-14)
dimethylglycine-Ser-piperazineacetic
C s Acm -Gl acid
L42 30-50%B5.651601.625 N,N- 4- 8-amino-3,6-none BBN(7-14)
dimethylglycine-Ser-Hydroxyprolinedioxaoctanoic
C s Acm -G1 acid
L48 30-50%B4.471600.540 N,N- 4-aminoproline8-amino-3,6-none BBN(7-
14)
dimethylglycine-Ser- dioxaoctanoic
CsAcm-Gl acid
L51 15-35%B5.141673.749 N,N- Lys 8-amino-3,6-Gly BBN(7-14)
dimethylglycine-Ser- dioxaoctanoic
C sAcm-Gl acid
L52 15-35%B6.081701.614 N,N- Arg 8-amino-3,6-Gly BBN(7-14)
dimethylglycine-Ser- dioxaoctanoic
C s Acm -GI acid
L53 15-35%B4.161632.610 N,N- Ser 8-amino-3,6-Gly BBN(7-14)
dimethylglycine-Ser- dioxaoctanoic
C s(Acm -Gl acid
CA 02549318 2006-06-08
WO 2005/067983 PCT/US2004/022115
31
'f
able
1
-
Compounds
Containing
Linkers
With
At
Least
One
Non-alpha
Amino
Acid
CompoHPLC HPLC
and method'RTZ MS3 IC505M N O P G*
L54 15-35%B4.881661.6>50 N,N- Asp 8-amino-3,6-Gly BBN(7-14)
dimethylglycine-Ser- dioxaoctanoic
C s Acm -Gl acid
L55 15-35%B4.831683.443 N,N- 8-amino-3,6-Asp Gly BBN(7-14)
(M+Na) dimethylglycine-Ser-dioxaoctanoic
C s(Acm)-GI acid
L56 IS-35%B4.651655.74 N,N- 8-amino-3,6-Ser Gly BBN(7-14)
(M+Na) dimethylglycine-Ser-dioxaoctanoic
Cys(Acm -Gly acid
L57 15-35%B4.9 1701.850 N,N- 8-amino-3,6-Arg Gly BBN(7-14)
dimethylglycine-Ser-dioxaoctanoic
C s Acm -GI acid
L58 15-35%B4.22846.4 >50 N,N- 8-amino-3,6-8-amino-3,6-Gly BBN(7-14)
(M+H/2) dimethylglycine-Ser-dioxaoctanoicdioxaoctanoic
C s Acm -GI acid acid
L59 15-35%B4.031635.542 N,N- 8-amino-3,6-2,3- Gly BBN(7-14)
dimethylgIycine-Ser-dioxaoctanoicdiaminopropio
C s Acm -GI acid nic acid
L60 IS-35%B4.111696.620 N,N- 8-amino-3,6-Lys Gly BBN(7-14)
(M+Na) dimethylglycine-Ser-dioxaoctanoic
C s(Acm -GI acid
L61 15-35%B4.321631.443 N,N- 2,3- 8-amino-3,6-Gly BBN(7-14)
dimethylglycine-Ser-diaminopropionidioxaoctanoic
C s Acm -Gl c acid acid
L78 20-40%B6.131691.435 D03A-monoamide8-amino-3,6-Diaminopropinone BBN(7-14)
(M+Na) dioxaoctanoiconic acid
acid
L79 20-40%B7.721716.842 D03A-monoamide8-amino-3,6-Biphenylalaninone BBN(7-
14)
(M+Na) dioxaoctanoicne
acid
L80 20-40%B7.781695.9>50 D03A-monoamide8-amino-3,6-Diphenylalaninone BBN(7-
14)
dioxaoctanoicne
acid
L81 20-40%B7.571513.637.5D03A-monoamide8-amino-3,6-4- none BBN(7-14)
dioxaoctanoicBenzoylpheny
acid lalanine
L92 I S-30%B5.631571.65 D03A-monoamide5- 8-amino-3,6-none BBN(7-14)
aminopentanoicdioxaoctanoic
acid ~ acid
L94 20-36%B4.191640.86.2 D03A-monoamide8-amino-3,6-D- none BBN(7-14)
(M+Na) dioxaoctanoicPhenylalanine
acid
L110 15-45%B5.061612.736 DO3A-monoamide8-aminooctanoic8-amino-3,6-none BBN(7-
14)
acid dioxaoctanoic
acid
L209 20-40%B4.623072.5437 D03A-monoamideE(G8-amino-8- 8- BBN(7-14)
over 3,6- aminooctanoicaminoo
6
minutes dioxaoctanoicacid ctanoic
acid-8-amino- acid
3,6-
dioxaoctanoic
acid
QWAVGHLM-
NHZ
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32
Table
B
-
Compounds
Containing
Linkers
With
At
Least
One
Non-alpha
Amino
Acid
CompoHPLC HPLC
and method'RTZ MS3 IC505M N O P G*
L210 20-50%B6.183056.76l D03A-monoamideE(G-Aoa-Aoa-8- 8- BBN(7-14)
I
over QWAVGHLM-aminooctanoicaminoo
minutes NI-IZ) acid ctanoic
acid
*BBN(7-14) is [SEQ ID NO:1
' HPLC method refers to the 10 minute time for the HPLC gradient.
z HPLC RT refers to the retention time of the compound in the HPLC.
3 MS refers to mass spectra where molecular weight is calculated from
mass/unit charge
5 (m/e).
4 ICso refers to the concentration of compound to inhibit 50% binding of
iodinated bombesin
to a GRP receptor on cells.
2B. Linkers Containing At Least One Substituted Bile Acid
[00159] In another embodiment of the present invention, the linker N-O-P
contains at least
10 one substituted bile acid. Thus, in this embodiment of the linker N-O-P,
N is 0 (where 0 means it is absent), an alpha amino acid, a substituted
bile acid or other linking group;
O is an alpha amino acid or a substituted bile acid; and
P is 0, an alpha amino acid, a substituted bile acid or other linking
group,
wherein at least one of N, O or P is a substituted acid.
[00160] Bile acids are found in bile (a secretion of the liver) and are
steroids having a
hydroxyl group and a five carbon atom side chain terminating in a carboxyl
group. In
substituted bile acids, at least one atom such as a hydrogen atom of the bile
acid is substituted
with another atom, molecule or chemical group. For example, substituted bile
acids include
those having a 3-amino, 24-carboxyl function optionally substituted at
positions 7 and 12
with hydrogen, hydroxyl or keto functionality.
[00161 ] Other useful substituted bile acids in the present invention include
substituted
cholic acids and derivatives thereof. Specific substituted cholic acid
derivatives include:
(313,513)-3-aminocholan-24-oic acid;
CA 02549318 2006-06-08
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33
(313,513,12a)-3-amino-12-hydroxycholan-24-oic acid;
(313,513,7a,12a)-3-amino-7,12-dihydroxycholan-24-oic acid;
Lys-(3,6,9)-trioxaundecane-1,11-dicarbonyl-3,7-dideoxy-3
aminocholic acid);
(313,513,7a)-3-amino-7-hydroxy-12-oxocholan-24-oic acid; and
(313,513,7a)-3-amino-7-hydroxycholan-24-oic acid.
[00162 Examples of compounds having the formula M-N-O-P-G which contain
linkers
with at least one substituted bile acid are listed in Table 2. These compounds
may be
prepared using the methods disclosed herein, particularly in the Examples, as
well as by
similar methods known to one skilled in the art.
TABLE 2
Table
2
-
Compounds
Containing
Linkers
With
At
Least
One
Substituted
Bile
Acid
CompoHPLC HPLC
and method'RTz MS3 IC505M N O P G*
L62 20- 3.79 1741.2>50 D03A-monoamideGly (313,513)-3-aminocholan-none
BBN(7-14)
80%B 24-oic acid
L63 20- 3.47 1757.023 D03A-monoamideGly (313,5J3,12a)-3-amino-12-none
BBN(7-14)
80%B hydroxycholan-24-oic
acid
L64 20- 5.31 1773.78.5 D03A-monoamideGly (313,513,7a,12a)-3-amino-none
BBN(7-14)
50%B 7,12-dihydroxycholan-
24-oic acid
L65 20- 3.57 2246.2>50 D03A-monoamideGly Lys-(3,6,9- Arg BBN(7-14)
80%B trioxaundecane-
l , l l -
dicarbonyl-3,7-
dideoxy-3-aminocholic
acid
L66 20-80%3.79 2245.8>50 D03A-monoamideGly Lys-(313,513,7a,12a)-3-Arg
BBN(7-14)
amino-7,12-
dihydroxycholan-24-oic
acid-3,6,9-
trioxaundecane-1,11-
dicarbon 1
L67 20-80%3.25 1756.94.5 D03A-monoamideGly (313,513,?a,l2a)-3-amino-none
BBN(7-14)
12-oxacholan-24-oic
acid
L69 20-80%3.25 1861.278 D03A-monoamide1-amino-(313,513,7a,12a)-3-amino-
none BBN(7-14)
3,6- 7,12-dihydroxycholan-
dioxaocta24-oic acid
noic
acid
L280 - - - - D03A-monoamideGly 3f3,5l37a,12a)-3-amino-none Q-W-A-
V-
7,12-dihydroxycholan- a-H-L-M-
24-oic acid NHz
L281 - - - - D03A-monoamideGly 313,513 7a,12a)-3-amino-f Q-W-A-
V-
7,12-dihydroxycholan- G-H-L-M-
24-oic acid NHz
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34
Table
2
-
Compounds
Containing
Linkers
With
At
Least
One
Substituted
Bile
Acid
CompoHPLC HPLC
and method'RTZ MS3 IC505M N O P G*
L282 - - - - D03A-monoamideGly 313,513 7a,12a)-3-amino-f Q-W-
A-V-
7,12-dihydroxycholan- G-H-L-L-
24-oic acid NHZ
L283 - - - - D03A-monoamideGly 3J3,SJ3 7a,12a)-3-amino-f Q-W-
A-V-
7,12-dihydroxycholan- G-H-L-
24-oic acid NH-pentyl
L284 - - - - D03A-monoamideGly 313,513 7a,12a)-3-amino-y
QWAVBal
7,12-dihydroxycholan- a-HFNle-
24-oic acid NHZ
L285 - - - - D03A-monoamideGly 313,513 7a,12a)-3-amino-f Q-W-
A-V-
7,12-dihydroxycholan- Bala-H-F-
24-oic acid Nle-NHZ
L286 - - - - D03A-monoamideGly 3J3,513 7a,12a)-3-amino-none
QWAVGH
7,12-dihydroxycholan- FL-NHZ
24-oic acid
L287 - - - - D03A-monoamideGly 313,513 7a,12a)-3-amino-none
QWAVGN
7,12-dihydroxycholan- MeHis-
24-oic acid LM-NHZ
L288 - - - - D03A-monoamideGly 313,513 7a,12a)-3-amino-none
LWAVGS
7,12-dihydroxycholan- F-M-NHZ
24-oic acid
L289 - - - - D03A-rnonoamideGly 313,513 7a,12a)-3-amino-none
HWAVGH
7,12-dihydroxycholan- L-M-NHZ
24-oic acid
L290 - - - - D03A-monoamideGly 313,513 7a,12a)-3-amino-none
LWATGH
7,12-dihydroxycholan- -F-M-NHZ
24-oic acid
L291 - - - - D03A-monoamideGly 3J3,513 7a,12a)-3-amino-none
QWAVGH
7,12-dihydroxycholan- -FMNHZ
24-oic acid
L292 - - - - D03A-monoamideGly 3J3,513 7a,12a)-3-amino-QRLG
QWAVGH
7,12-dihydroxycholan-N LM-NHz
24-oic acid
L293 - - - - D03A-monoamideGly 313,513 7a,12a)-3-amino-QRYG
QWAVGH
7,12-dihydroxycholan-N LM-NHZ
24-oic acid
L294 - - - - D03A-monoamideGly 313,513 7a,12a)-3-amino-QKYG
QWAVGH
7,12-dihydroxycholan-N LM-NHZ
24-oic acid
L295 - - - - Pglu-Q-Lys Gly 313,513 7a,12a)-3-amino-LG-N QWAVGH
(D03A- 7,12-dihydroxycholan- LM-NHZ
monoamide) 24-oic acid
L303 D03A-monoamideGly 3-amino-3-deoxycholicnone QRLGNQ
- - - - acid WAVGHL
M-NHZ
L304 D03A-monoamideGly 3-amino-3-deoxycholicnone QRYGNQ
- - - - acid WAVGHL
M-NHZ
L305 D03A-monoamideGly 3-amino-3-deoxycholicnone QKYGNQ
- - - - acid WAVGHL
M_NHZ
CA 02549318 2006-06-08
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'f
able
2
-
Compounds
Containing
Linkers
With
At
Least
One
Substituted
Bile
Acid
Compo-I-IPLCIIPLC
and method'I2T2 MS3 IC505M N O P G
L306 D03A-monoamideGly 3-amino-3-deoxycholicnone See
FIG.
- - - - acid 38 for
structure
of
targeting
a tide
*BBN(7-14) is [SEQ ID NO:1]
HPLC method refers to the 10 minute time for the HPLC gradient.
~ HPLC RT refers to the retention time of the compound in the HPLC.
3 MS refers to mass spectra where molecular weight is calculated from
masslunit charge
5 (m1e).
4 ICSO refers to the concentration of compound to inhibit 50% binding of
iodinated bombesin
to a GRP receptor on cells.
2C. Linkers Containing At Least One Non-Alpha Amino Acid With A Cyclic Group
[00163] In yet another embodiment of the present invention, the linker N-O-P
contains at
10 least one non-alpha amino acid with a cyclic group. Thus, in this
embodiment of the linker
N-O-P,
N is 0 (where 0 means it is absent), an alpha amino acid, a non-alpha
amino acid with a cyclic group or other linking group;
O is an alpha amino acid or a non-alpha amino acid with a cyclic
15 group; and
P is 0, an alpha amino acid, a non-alpha amino acid with a cyclic
group, or other linking group,
wherein at least one of N, O or P is a non-alpha amino acid with a
cyclic group.
20 [00164] Non-alpha amino acids with a cyclic group include substituted
phenyl, biphenyl,
cyclohexyl or other amine and carboxyl containing cyclic aliphatic or
heterocyclic moieties.
Examples of such include:
4-aminobenzoic acid (hereinafter referred to as "Abz4 in the
specification")
3-aminobenzoic acid
4-aminomethyl benzoic acid
CA 02549318 2006-06-08
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36
8-aminooctanoic acid
trans-4-aminomethylcyclohexane carboxylic acid
4-(2-aminoethoxy)benzoic acid
isonipecotic acid
2-aminomethylbenzoic acid
4-amino-3-nitrobenzoic acid
4-(3-carboxymethyl-2-keto-1-benzimidazolyl-piperidine
6-(piperazin-1-yl)-4-(3H)-quinazolinone-3-acetic acid
(25,5 S)-5-amino-1,2,4,5,6,7-hexahydro-
azepino[3,21-hi]indole-4-one-2-carboxylic acid
(4S,7R)-4-amino-6-aza-5-oxo-9-thiabicyclo[4.3.0]nonane-7-
carboxylic acid
3-carboxymethyl-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one
N1-piperazineacetic acid
N-4-aminoethyl-N-1-piperazineacetic acid
(3 S)-3-amino-1-carboxymethylcaprolactam
(2S,6S,9)-6-amino-2-carboxymethyl-
3,8-diazabicyclo-[4,3,0]-nonane-1,4-dione
3-amino-3-deoxycholic acid
4-hydroxybenzoic acid
4-aminophenylacetic acid
3-hydroxy-4-aminobenzoic acid
3-methyl-4-aminobenzoic acid
3-chloro-4-aminobenzoic acid
3-methoxy-4-aminobenzoic acid
6-aminonaphthoic acid
N,N'-Bis(2-aminoethyl)-succinamic acid
[00165] Examples of compounds having the formula M-N-O-P-G which contain
linkers
with at least one alpha amino acid with a cyclic group are listed in Table 3.
These
compounds may be prepared using the methods disclosed herein, particularly in
the
Examples, as well as by similar methods known to one skilled in the art.
CA 02549318 2006-06-08
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37
TABLE 3
Table
3
-
Compounds
Containing
Linkers
Related
To
Amino-(Phenyl,
Biphenyl,
Cycloalkyl
Or
Heterocyclic)
Carboxylates
Comp HPLC HPLC
ound method'RTZ MS3 ICSOSM N O p G*
L70 10-40%B6.15 1502.65 DO3A- Gly 4-aminobenzoicnone BBN(7-14)
acid
monoamide
L71 20-50%14.14 59.68 7 D03A- none 4-aminomethylnone BBN(7-14)
over (M+Na) monoamide benzoic acid
30
minutes
L72 20-50%13.64 65.73 8 D03A- none trans-4- none BBN(7-14)
over (M+K) monoamide aminomethylcyclohe
30
minutes x 1 carbox
lic acid
L73 5-35%7.01 1489.85 D03A- none 4-(2- none BBN(7-14)
monoamide aminoethoxy)benzoic
acid
L74 S-35%6.49 1494.87 D03A- Gly isonipecoticnone BBN(7-14)
acid
monoamide
L75 5-35%6.96 1458.023 D03A- none 2- none BBN(7-14)
monoamide aminomethylbenzoic
acid
L76 5-35%7.20] 1502.74 DO3A- none 4-aminomethyl-3-none BBN(7-14)
monoamide nitrobenzoic
acid
L77 20-40%B6.17 1691.817.5D03A- 8-amino-1-Naphthylalaninenone BBN(7-14)
(M+Na) monoamide3,6-
dioxaoctan
oic
acid
L82 20-40%B6.18 1584.68 D03A- none 4-(3-carboxymethyl-none BBN(7-
14)
monoamide 2-keto-1-
benzimidazolyl-
i eridine
L83 20-40%B5.66 1597.5>50 D03A- none 6-(piperazin-I-yl)-4-none BBN(7-
14)
monoamide (3H)-quinazolinone-
3-acetic
acid
L84 20-40%B6.31 1555.5>50 D03A- none (25,55)-5-amino-none BBN(7-14)
monoamide 1,2,4,5,6,7-
hexahydro-
azepino[3,21-
hi]indole-
4-one-2-carboxylic
acid
L85 20-40%B5.92 1525.5>50 D03A- none (4S,7R)-4-amino-6-none BBN(7-14)
monoamide aza-5-oxo-9-
thiabicyclo[4.3.0]non
ane-7-carbox
lic acid
L86 20-40%B6.46 1598.6>50 D03A- none N,N-dimethylglycinenone BBN(7-
14)
monoamide
L87 20-40%B5.47 1593.8>50 D03A- none 3-carboxymethyl-1-none BBN(7-14)
(M+Na) monoamide phenyl-1,3,8-
t riazaspiro[4.5]decan
- 4-one
L88 20-40%B3.84 1452.7>50 D03A- none NI-piperazineaceticnone BBN(7-
14)
monoamidea cid
CA 02549318 2006-06-08
WO 2005/067983 PCT/US2004/022115
38
't'able
3
-
Compounds
Containing
Linkers
Related
To
Amino-(Phenyl,
Biphenyl,
Cycloalkyl
Or
Heterocyclic)
Carboxylates
CompHPLC HPLC
oundmethod'RTZ MS3 IC505M N O P G*
L89 20-40%B5.68 1518.523 D03A- none N-4-aminoethyl-N-1-none BBN(7-
14)
(M+Na) monoamide piperazine-acetic
acid
L90 20-40%B7.95 1495.450 D03A- none (3S)-3-amino-1-none BBN(7-14)
monoamide carboxymethylcaprol
actam
L91 20-40%B3.97 1535.7>50 D03A- none (2S,6S,9)-6-amino-2-none BBN(7-
14)
monoamide carboxymethyl-3,8-
diazabicyclo-
[4,3,0]-nonane-1,4-
dione
L93 15-30%B7.57 1564.75.8 D03A- 5- trans-4- none BBN(7-14)
monoamideaminopentaminomethylcyclohe
anoic xane-I-carboxylic
acid
acid
L95 15-35%B5.41 1604.614 D03A- trans-4-D-Phenylalaninenone BBN(7-14)
monoamideaminometh
ylcyclohex
ane-1-
carboxylic
acid
L96 20-36%B4.75 1612.735 DO3A- 4- 8-amino-3,6-none BBN(7-14)
monoamideaminomethdioxaoctanoic
acid
ylbenzoic
acid
L97 15-35%B5.86 1598.84.5 D03A- 4-benzoyl-trans-4- none BBN(7-14)
monoamide(L)- aminomethylcyclohe
phenylalanxane-1-carboxylic
ine acid
L98 15-35%B4.26 1622.716 D03A- trans-4-Arg none BBN(7-14)
monoarnideaminometh
ylcyclohex
ane-1-
carboxylic
acid
L99 15-35%B4.1 1594.722 D03A- trans-4-Lys none BBN(7-14)
monoamideaminometh
ylcyclohex
ane-1-
carboxylic
acid
L10015-35%B4.18 1613.610 D03A- trans-4-Diphenylalaninenone BBN(7-14)
monoamideaminometh
ylcyclohex
ane-1-
carboxylic
acid
L10115-35fB5.25 1536.725 D03A- trans-4-1-Naphthylalaninenone BBN(7-
14)
monoamideaminometh
ylcyclohex
ane-1-
carboxylic
acid
CA 02549318 2006-06-08
WO 2005/067983 PCT/US2004/022115
39
Table
3
-
Compounds
Containing
Linkers
Related
To
Amino-(Phenyl,
Biphenyl,
Cycloalkyl
Or
Heterocyclic)
Carboxylates
CompHPLC HPLC
oundmethod'RTZ MS's IC505M N O P G*
L10215-35%B5.28 1610.89.5 D03A- traps-4-8-amino-3,6-none BBN(7-14)
monoamideaminomethdioxaoctanoic
acid
ylcyclohex
ape-1-
carboxylic
acid
L10315-35%B4.75 1552.724 D03A- traps-4-Ser none BBN(7-14)
monoamideaminometh
ylcyclohex
ape-1-
carboxylic
acid
L10415-35%B3.91 1551.732 D03A- traps-4-2,3-diaminopropionicnone BBN(7-
14)
monoamideaminomethacid
ylcyclohex
ape-I-
carboxylic
acid
L10520-45%B7.68 1689.73.5 D03A- traps-4-Biphenylalaninenone BBN(7-14)
monoamideaminometh
ylcyclohex
ape-1-
carboxylic
acid
L10620-45%B6.97 1662.73.8 D03A- traps-4-(2S,5S)-5-amino-none BBN(7-14)
monoamideaminometh1,2,4,5,6,7-
ylcyclohexhexahydro-
ane-I-azepino[3,21-
carboxylichi]indole-
acid 4-one-2-carboxylic
acid
L10715-35%B5.79 1604.75 DO3A- traps-4-traps-4- none BBN(7-14)
monoamideaminomethaminomethylcyclohe
ylcyclohexxane-1-carboxylic
ape-1-acid
carboxylic
acid
L10815-45%B6.38 1618.710 D03A- 8-amino-Phenylalaninenone BBN(7-14)
monoamide3,6-
dioxaoctan
oic
acid
Ll 15-45%B6.85 1612.76 DO3A- traps-4-Phenylalaninenone BBN(7-14)
09
monoamideaminometh
ylcyclohex
ape-1-
carboxylic
acid
L1 20-45%B3.75 1628.68 D03A- 8- raps-4- none BBN(7-14)
11 t
monoamideaminooctaaminomethylcyclohe
noic xane-1-carboxylic
acid
acid
Ll 20-47%B3.6 1536.54.5 D03A- none 4'-aminomethyl-none BBN(7-14)
12
in monoamide biphenyl-1-
9
min
carbox lic
acid
CA 02549318 2006-06-08
WO 2005/067983 PCT/US2004/022115
Table
3
-
Compounds
Containing
Linkers
Related
To
Amino-(Phenyl,
Biphenyl,
Cycloalkyl
Or
Heterocyclic)
Carboxylates
CompHPLC HPLC
oundmethod'RTZ MS3 IC505M N O P G*
L11320-47%B3.88 1558.65 DO3A- none 3'-aminomethyl-none BBN(7-14)
in (M+Na) monoamide biphenyl-3-
9
min
carbox lic
acid
L11410-40%B5.47 1582.84.5 CMDOTA Gl 4-aminobenzoicnone BBN(7-14
acid
L1245-35%B7.04 1489.98.0 D03A- none 4- none BBN(7-14)
monoamide aminomethylphenox
acetic acid
L1435-35%B6.85 1516.8I1 D03A- Gly 4-aminophenylaceticnone BBN(7-14)
monoamide acid
L1445-35%B6.85 1462.79 HPD03A none 4- henox none BBN(7-14
L14520-80%B1.58 1459.85 D03A- none 3- none BBN(7-14)
monoamide aminomethylbenzoic
acid
L14620-80%B1.53 1473.79 D03A- none 4- none BBN(7-14)
monoamide aminomethylphenyla
cetic acid
L14720-80%B1.68 1489.73.5 D03A- none 4-aminomethyl-3-none BBN(7-14)
monoamide methox benzoic
acid
L20110-46%B5.77 1563.736 Boa*** none Gly 4- BBN(7-14)
over aminoben
12
minutes zoic
acid
L20210-46%B5.68 1517.7413 D03A- none Gly 4- BBN(7-14)
over monoamide hydrazino
12
minutes benzo
1
L20310-46%B5.98 1444.699 D03A- none none 4- BBN(7-14)
over monoamide aminoben
12
minutes zoic
acid
L20410-46%B5.82 1502.7350 D03A- none 4-aminobenzoicGly BBN(7-14)
acid
over monoamide
12
minutes
L20510-46%B5.36 1503.7245 D03A- Gly 6-Aminonicotinicnone BBN(7-14)
over monoamide acid
12
minutes
L20610-46%B7.08 1592.854.5 D03A- Gly 4'-Amino-2'-methylnone BBN(7-14)
over monoamide biphenyl-4-
12
minutes carbox lic
acid
L20710-46%B7.59 1578.832.5 D03A- Gly 3'-Aminobiphenyl-3-none BBN(7-
14)
over monoamide carboxylic
12 acid
minutes
L20810-46%B5.9 1516.757.5 D03A- Gly 1,2-diaminoethylTerephthaBBN(7-
14)
over monoamide lic
12 acid
minutes
L21110-46JB5.76 1560.774 D03A- Gly Gly 4- BBN(7-14)
over monoamide aminoben
12
minutes zoic
acid
L21210-46!B6.05 1503.71NT** D03A- none Gly 4- EWAVGH
over monoamide aminobenLM-NHZ
12
minutes . zoic
acid
L21310-46fB5.93 1503.71NT** D03A- Gly 4-aminobenzoicnone QWAVGH
acid
over monoamide LM-OH
12
minutes
CA 02549318 2006-06-08
WO 2005/067983 PCT/US2004/022115
41
Table
3
-
Compounds
Containing
Linkers
Related
To
Amino-(Phenyl,
Biphenyl,
Cycloalkyl
Or
Heterocyclic)
Carboxylates
Comp HPLC HPLC
ound method'RTZ MS3 IC505M N O P G*
L2I4 10-46%B7.36 1649.91NT** D03A- Gly 4-aminobenzoic(D)-PheBBN(7-14)
acid
over monoamide
12
minutes
L215 10-46%B5.08 2071.37NT** D03A- Gly 4-aminobenzoicnone QRLGNQ
acid
over monoamide WAVGHL
12
minutes M-NHz
L216 10-46%B4.94 2121.38NT** DO3A- Gly 4-aminobenzoicnone QRYGNQ
acid
over monoamide WAVGHL
12
minutes
M-NH2
L217 10-46%B4.38 2093.37NT** DO3A- Gly 4-aminobenzoicnone QKYGNQ
acid
over monoamide WAVGHL
12
minutes ' M-NH2
L218 10-46%B6.13 2154.45NT** DO3A- Gly 4-aminobenzoicnone See
acid FIG.
over monoamide 38 for
12
minutes structure
of
targeting
a tide
L219 10-46%B8.61 1588.84NT** DO3A- Gly 4-aminobenzoic(D)-PheQWAVGH
acid
over monoamide L-NH-
12
minutes Pent
1
L220 10-46%B5.96 1516.75NT** D03A- Gly 4-aminobenzoicnone QWSVaH
acid
over monoamide LM-NHZ
12
minutes
L221 10-46%B7.96 1631.87NT** D03A- Gly 4-aminobenzoic(D)-PheQWAVGH
acid
over monoamide LL-NHZ
12
minutes
L222 10-46%B6.61 1695.91NT** DO3A- Gly 4-aminobenzoic(D)-TyrQWAV-
acid
over monoamide Bala-HF-
12
minutes Nle-NH
L223 10-46%B7.48 1679.91NT** D03A- Gly 4-aminobenzoicPhe QWAV-
acid
over monoamide Bala-HF-
12
minutes Nle-NHz
L224 10-46%B5.40 1419.57NT** D03A- Gly 4-aminobenzoicnone QWAGHF
acid
over monoamide L,-NHz
12
minutes
L225 10-46%B8.27 1471.71NT** D03A- Gly 4-arninobenzoicnone LWAVGS
acid
over monoamide FM-NH2
12
minutes
L226 10-46%B5.12 1523.75NT** D03A- Gly 4-aminobenzoicnone HWAVGH
acid
over monoamide LM-NH2
12
minutes
L227 10-46%B6.61 1523.75NT** D03A- Gly 4-aminobenzoienone LWAVGS
acid
over monoamide FM-NH2
12
minutes
L228 10-46%B5.77 1511 NT** D03A- Gly 4-aminobenzoicnone QWAVGH
acid
over monoamide FM-NHZ
12
minutes
L233 5-35%B7.04 1502.714.8 D03A- Gly 3-aminobenzoicnone BBN(7-14)
acid
over monoamide
min
L234 20-80%1.95 1552.763 D03A- Gly 6-aminonaphthoicnone BBN(7-14)
over monoamide acid
10
minutes
CA 02549318 2006-06-08
WO 2005/067983 PCT/US2004/022115
42
Table
3
-
Compounds
Containing
Linkers
Related
To
Amino-(Phenyl,
Biphenyl,
Cycloalkyl
Or
Heterocyclic)
Carboxylates
Comp HPLC HPLC
ound method'RTZ MS3 IC505M N O P G~
L235 20-80%1.95 1515.72? D03A- Gly 4- none BBN(7-14)
over monoamide methylaminobenzoic
minutes acid
L237 20-80%1.52 1538.685 Cm4pmlOd2aGly 4-aminobenzoicnone BBN(7-14)
acid
over
10
minutes
L238 5-35%B7.17 1462.701.5 N,N- Gly 4-aminobenzoicnone BBN(7-14)
acid
over dimethylglycin
10
min e-Ser-
C s(Acm
-Gl
L239 20-80%3.36 1733.164.5 N,N- Gly 3-amino-3- none BBN(7-14)
over dimethylglycin deoxycholic
10 acid
minutes e-Ser-
C s(Acm
-Gl
L240 20-80%1.55 1532.734 D03A- Gly 3-methoxy-4-none BBN(7-14)
over monoamide aminobenzoic
10 acid
minutes
L241 20-80!1.63 1535.684 D03A- Gly 3-chloro-4- none BBN(7-14)
over monoamide aminobenzoic
10 acid
minutes
L242 20-80%1.55 1516.755 D03A- Gly 3-methyl-4- none BBN(7-14)
over monoamide aminobenzoic
10 acid
minutes
L243 20-80%1.57 1518.7014 D03A- Gly 3-hydroxy-4-none BBN(7-14)
over monoamide aminobenzoic
10 acid
minutes
L244 5-50% 4.61 1898.16>50 (D03A- N,N'- none none BBN(7-14)
over monoamide)2Bis(2-
10
minutes aminoethyl
)_
succinamic
acid
L300 10-46%- - - D03A- Gly 4-aminobenzoicnone QWAVGH
acid
over monoamide FL-NHz
10
minutes
L301 20-45%7.18 - - DO3A- none 4- L-I- BBN(7-14)
over monoamide aminomethylbenzoicNaphthyla
minutes acid lanine
L302 - - - - D03A- Gly 4-aminobenzoicnone QWAVGN
acid
monoamide MeHis-L-
M-NHa
*BBN(7-14) is [SEQ ID N0:1]
**NT is defined as "not tested."
~'**BOA is defined as (1R)-1-(Bis{2-
[bis(carboxymethyl)amino]ethyl~amino)propane-1,3-
dicarboxylic acid.
' HPLC method refers to the 10 minute time for the HPLC gradient.
Z HPLC RT refers to the retention time of the compound in the HPLC.
' MS refers to mass spectra where molecular weight is calculated from
mass/unit charge
(m/e).
CA 02549318 2006-06-08
WO 2005/067983 PCT/US2004/022115
43
4 ICSO refers to the concentration of compound to inhibit 50% binding of
iodinated bombesin
to a GRP receptor on cells.
[00166 A subset of compounds containing preferred linkers and various GRP
receptor
targeting peptides are set forth in Table 4. These compounds may be prepared
using the
methods disclosed herein, particularly in the Examples, as well as by similar
methods known
to one skilled in the art.
TABLE 4
Table
4
-
Compounds
Containing
Linkers
of
the
Invention
With
Various
GRP-R
Targeting
Moities
CompouHPLC HPLC
nd method'RTZ MS3 IC505 M N O P G*
L214 10-46%B7.36 1649.91NT** D03A- Gly 4-aminobenzoic(D)-BBN(7-14)
acid
over monoamide Phe
12
minutes
L215 10-46%B5.08 2071.37NT** D03A- GIy 4-aminobenzoicnoneQRLGNQWA
acid
over monoamide VGHLM-NHZ
12
minutes
L216 10-46%B4.94 2121.38NT** DO3A- Gly 4-aminobenzoicnoneQRYGNQWA
acid
over monoamide VGHLM-NHZ
12
minutes
L217 10-46%B4.38 2093.37NT** D03A- Gly 4-aminobenzoicnoneQKYGNQWA
acid
over monoamide VGHLM-NH2
12
minutes
L218 10-46%B6.13 2154.45NT** D03A- Gly 4-aminobenzoicnoneSee FIG.
acid 38
over monoamide for structure
12 o
minutes targeting
a tide
L219 10-46%B8.61 1588.84NT** D03A- Gly 4-aminobenzoic(D)-QWAVGHL-
acid
over monoamide Phe NH-Pentyl
12
minutes
L220 10-46%B5.96 1516.75NT** D03A- Gly 4-aminobenzoicnoneQWAVaHLM
over monoamide acid -NH2
12 minutes
L221 10-46%B7.96 1631.87NT** D03A- Gly 4-aminobenzoic(D)-QWAVGHLL
acid
over monoamide Phe -NHZ
12
minutes
L222 10-46%B6.61 1695.91NT** D03A- Gly 4-aminobenzoic(D)-QWAV-Bala-
acid
over monoamide Tyr HF-Nle-NHZ
12
minutes
L223 10-46%B7.48 1679.91NT** D03A- Gly 4-aminobenzoicPhe QWAV-Bala-
acid
over monoamide HF-Nle-NHZ
12
minutes
L224 10-46%B5.40 1419.57NT** D03A- Gly 4-aminobenzoicnoneQWAGHFL-
acid
over monoamide NHz
12
minutes
L225 10-46%B8.27 1471.71NT** D03A- Gly 4-aminobenzoicnoneLWAVGSFM
acid
over monoamide -NHa
12
minutes
L226 10-46%B5.12 1523.75NT** D03A- Gly 4-aminobenzoicnoneHWAVGHL
acid
over monoamide M-NHz
I2
minutes
CA 02549318 2006-06-08
WO 2005/067983 PCT/US2004/022115
44
Tahle
4
-
Compounds
Containing
Linkers
of
the
Invention
With
Various
GRP-R
Targeting
Moities
CornpouHPLC HPLC
nd method'RTZ MS3 IC505 M N O P G
L227 10-46%B6.61 1523.75NT** D03A- Gly 4-aminobenzoicnoneLWATGHFM
acid
over monoamide -NHz
12
minutes
L228 10-46%B5.77 1511 NT** D03A- Gly 4-aminobenzoicnoneQWAVGHFM
acid
over monoamide -NHz
12
minutes
L280 D03A- Gly (313,513 7a,12a)-3-noneQWAVaHLM
-- -- -- -- monoamide amino-7,12- -NHZ
dihydroxycholan-24-
oic acid
L281 D03A- Gly (313,513 7a,12a)-3-f QWAVGH-
-- -- -- -- monoamide amino-7,12- LM-NHz
dihydroxycholan-24-
oic acid
L282 D03A- Gly (313,573 ?a,l2a)-3-f QWAVGHLL
-- -- -- -- monoamide amino-7,12- -NHz
dihydroxycholan-24-
oic acid
L283 D03A- Gly (313,513 ?a,l2a)-3-f QWAVGHLN
-- -- -- -- monoamide amino-7,12- H-pentyl
dihydroxycholan-24-
oic acid
L284 D03A- Gly (313,5137a,12a)-3-y QWAVBaIaH
-- -- -- -- monoamide amino-7,12- F-Nle-NHz
dihydroxycholan-24-
oic acid
L285 D03A- Gly (3f3,513 7a,12a)-3-f QWAVBala-
-- -- -- -- monoamide amino-?,12- HF-Nle-NHz
dihydroxycholan-24-
oic acid
L286 D03A- Gly (313,513 7a,I2a)-3- QWAVGHFL
-- -- -- -- monoamide amino-7,12- none-NHz
dihydroxycholan-24-
oic acid
L287 DO3A- Gly (3I3,573 7a,12a)-3- QWAVGNMe
-- -- -- -- monoamide amino-7,12- noneHis-L-M-NHz
dihydroxycholan-24-
oic acid
L~$g D03A- Gly (313,513 7a,12a)-3- LWAVGSFM
-- -_ -- -- monoamide amino-7,12- none-NHz
dihydroxycholan-24-
oic acid
L289 DO3A- Gly (313,513 7a,12a)-3- HWAVGHL
-- -- -- -- monoamide amino-7,12- noneM-NHZ
dihydroxycholan-24-
oic acid
L290 D03A- Gly (373,513 7a,12a)-3- LWATGHFM
-- -- -- -- monoamide amino-?,12- none-NHZ
dihydroxycholan-24-
oic acid
L291 _ D03A- Gly (313,513 7a,12a)-3- QWAVGHFM
- - - - monoamide amino-7,12- none-NHz
dihydroxycholan-24-
oic acid
CA 02549318 2006-06-08
WO 2005/067983 PCT/US2004/022115
'fable
~
-
Compounds
Containing
Linkers
of
the
Invention
With
Various
GRP-R
Targeting
Moities
CompouHPLC HPLC
nd method'RTZ MS3 IC505 M N O P G*
L292 - - - - D03A- Gly 313,5J3 7a,12a)-3-QRLGQWAVGHL
monoamide amino-7,12- N M-NHZ
dihydroxycholan-24-
oic acid
L293 - - - - D03A- Gly 313,513 7a,12a)-3-QRYGQWAVGHL
monoamide amino-7,12- N M-NHZ
dihydroxycholan-24-
oic acid
L294 - - - - DO3A- Gly 313,513 7a,12a)-3-QKY QWAVGHL
monoamide amino-7,12- GN M-NHZ
dihydroxycholan-24-
oic acid
L295 - - - - Pglu-Q-LysGly 313,513 7a,12a)-3-LG-NQWAVGHL
(D03A- amino-7,12- M-NHZ
monoamide) dihydroxycholan-24-
oic acid
L304 D03A- Gly 3-amino-3- noneQRYGNQWA
- - - - monoamide deox cholic VGHLM-NHZ
acid
L305 D03A- Gly 3-amino-3- noneQKYGNQWA
- - - - monoamide deox cholic VGHLM-NH2
acid
L306 D03A- Gly 3-amino-3- noneSee FIG.
38
monoamide deoxycholic for structure
acid o
targeting
a tide
2D. Other Linking Groups
[00167] Other linking goups which may be used within the linker N-O-P include
a
chemical group that serves to couple the GRP receptor targeting peptide to the
metal chelator
or optical label while not adversely affecting either the targeting function
of the GRP receptor
targeting peptide or the metal complexing function of the metal chelator or
the detectability
of the optical label. Suitable other linking groups include peptides (i.e.,
amino acids linked
together) alone, a non-peptide group (e.g., hydrocarbon chain) or a
combination of an amino
acid sequence and a non-peptide spacer.
10 [00168] In one embodiment, other linking groups for use within the linker N-
O-P include
L-glutamine and hydrocarbon chains, or a combination thereof.
[00I 69] In another embodiment, other linking groups for use within the linker
N-O-P
include a pure peptide linking group consisting of a series of amino acids
(e.g., diglycine,
triglycine, gly-gly-glu, gly-ser-gly, etc.), in which the total number of
atoms between the N-
CA 02549318 2006-06-08
WO 2005/067983 PCT/US2004/022115
46
terminal residue of the GRP receptor targeting peptide and the metal chelator
or the optical
label in the polymeric chain is s 12 atoms.
[00170) In yet a further embodiment, other linking groups for use within the
linker N-O-P
can also include a hydrocarbon chain [i.e., R~-(CHZ)"-R2] wherein n is 0-10,
preferably n = 3
to 9, R~ is a group (e.g., H2N-, HS-, -COOH) that can be used as a site for
covalently linking
the ligand backbone or the prefonned metal chelator or metal complexing
backbone or
optical label; and R2 is a group that is used for covalent coupling to the N-
terminal NH2-
group of the GRP receptor targeting peptide (e.g., R2 is an activated COOH
group). Several
chemical methods for conjugating ligands (i.e., chelators) or preferred metal
chelates to
biomolecules have been well described in the literature [Wilbur, 1992; Parker,
1990;
Hermanson, 1996; Frizberg et aL, 1995]. One or more of these methods could be
used to link
either the uncomplexed ligand (chelator) or the radiometal chelate or optical
label to the
linker or to link the linker to the GRP receptor targeting peptides. These
methods include the
formation of acid anhydrides, aldehydes, arylisothiocyanates, activated
esters, or N-
hydroxysuccinimides [Wilbur, 1992; Parker, 1990; Hermanson, 1996; Frizberg et
al., 1995].
[00171) In a preferred embodiment, other linking groups for use within the
linker N-O-P
may be formed from linker precursors having electrophiles or nucleophiles as
set forth below:
LP 1: a linker precursor having on at least two locations of the linker
the same electrophile El or the same nucleophile Nul;
LP2: a linker precursor having an electrophile E1 and on another
location of the linker a different electrophile E2;
LP3: a linker precursor having a nucleophile Nul and on another
location of the linker a different nucleophile Nu2; or
LP4: a linker precursor having one end functionalized with an
electrophile El and the other with a nucleophile Nul.
[00172] The preferred nucleophiles Nul/Nu2 include-OH, -NH, -NR, -SH, -HN-NHa,
-
RN-NH2, and -RN-NHR', in which R' and R are independently selected from the
definitions
for R given above, but for R' is not H.
[00173] The preferred electrophiles El/E2 include -COOH, -CH=O (aldehyde), -
CR=OR'
(ketone), -RN-C=S, -RN-C=O,-S-S-2-pyridyl, -SOZ-Y, -CHaC(=O)Y , and
CA 02549318 2006-06-08
WO 2005/067983 PCT/US2004/022115
47
O ~N. O~~N~ O
' '
wherein Y can be selected from the following groups:
N-N=N CI, Br, F
O
N
N
~O_N N\ ~ \ N N ~ \ N~N~ <N N'
'N N ~ \\ _ N N -~ ~N- N
I I
O .r"-0 ,,,~-O
F CI NO~
0 \ F ,~O \ CI J~O \. f"'wO
l
F / F CI / CI / N02 I / NO
F CI
O
O ~ .~,
O O~I I \ ~ O \ S S
N~ /
N N / N-N
F
'~,S '~S~S I ~ NC / F ~ F
N / I
"~., S wN ~ ~-.S / F
F
~O
S N
GRP receptor tar etin -~~eptide
[00174] The GRP receptor targeting peptide (i. e., G in the formula M-N-O-P-G)
is any
peptide, equivalent, derivative or analogue thereof which has a binding
affinity for the GRP
receptor family.
[00175] The GRP receptor targeting peptide may take the form of an agonist or
an
antagonist. A GRP receptor targeting peptide agonist is known to "activate"
the cell
CA 02549318 2006-06-08
WO 2005/067983 PCT/US2004/022115
48
following binding with high affinity and may be internalized by the cell.
Conversely, GRP
receptor targeting peptide antagonists are known to bind only to the GRP
receptor on the cell
without being internalized by the cell and without "activating" the cell. In a
preferred
embodiment, the GRP receptor targeting peptide is an agonist.
[00176] In a more preferred embodiment of the present invention, the GRP
agonist is a
bombesin (BBN) analogue and/or a derivative thereof. The BBN derivative or
analog thereof
preferably contains either the same primary structure of the BBN binding
region (i.e.,
BBN(7-14) [SEQ ID NO:l]) or similar primary structures, with specific amino
acid
substitutions that will specifically bind to GRP receptors with better or
similar binding
affinities as BBN alone (i.e., I~d<25nM). Suitable compounds include peptides,
peptidomimetics and analogues and derivatives thereof. The presence of L-
methionine (Met)
at position BBN-14 will generally confer agonistic properties while the
absence of this
residue at BBN-14 generally confers antagonistic properties [Hoffken, 1994].
Some useful
bombesin analogues are disclosed in U.S. Patent Pub. 2003/0224998,
incorporated here in its
entirety.
[00177] It is well documented in the art that there are a few and selective
number of
specific amino acid substitutions in the BBN (8-14) binding region (e.g., D-
Alal' for L-Gly"
or D-Trp$ for L-TrpB), which can be made without decreasing binding affinity
[Leban et al.,
1994; Qin et al., 1994; Jensen et al., 1993]. In addition, attachment of some
amino acid
chains or other groups to the N-terminal amine group at position BBN-8 (i. e.,
the TrpB
residue) can dramatically decrease the binding affinity of BBN analogues to
GRP receptors
[Davis et al., 1992; Hoffken, 1994; Moody et al., 1996; Coy, et al., 1988; Cai
et al., 1994]. In
a few cases, it is possible to append additional amino acids or chemical
moieties without
decreasing binding affinity.
[00178] Analogues of BBN receptor targeting peptides include molecules that
target the
GRP receptors with avidity that is greater than or equal to BBN, as well as
muteins,
retropeptides and retro-inverso-peptides of GRP or BBN. One of ordinary skill
will
appreciate that these analogues may also contain modifications which include
substitutions,
and/or deletions and/or additions of one or several amino acids, insofar that
these
modifications do not negatively alter the biological activity of the peptides
described therein.
These substitutions may be carried out by replacing one or more amino acids by
their
synonymous amino acids. Synonymous amino acids within a group are defined as
amino
CA 02549318 2006-06-08
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49
acids that have sufficient physicochemical properties to allow substitution
between members
of a group in order to preserve the biological function of the molecule.
[00179] Deletions or insertions of amino acids may also be introduced into the
defined
sequences provided they do not alter the biological functions of said
sequences.
Preferentially such insertions or deletions should be limited to l, 2, 3, 4 or
S amino acids and
should not remove or physically disturb or displace amino acids which are
critical to the
functional conformation. Muteins of the GRP receptor targeting peptides
described herein
may have a sequence homologous to the sequence disclosed in the present
specification in
which amino acid substitutions, deletions, or insertions are present at one or
more amino acid
positions. Muteins may have a biological activity that is at least 40%,
preferably at least
50%, more preferably 60-70%, most preferably 80-90% of the peptides described
herein.
However, they may also have a biological activity greater than the peptides
specifically
exemplified, and thus do not necessarily have to be identical to the
biological function of the
exemplified peptides. Analogues of GRP receptor targeting peptides also
include
peptidomimetics or pseudopeptides incorporating changes to the amide bonds of
the peptide
backbone, including thioamides, methylene amines, and E-olefins. Also peptides
based on
the structure of GRP, BBN or their peptide analogues with amino acids replaced
by N-
substituted hydrazine carbonyl compounds (also known as aza amino acids) are
included in
the term analogues as used herein.
[00180] The GRP receptor targeting peptide can be prepared by various methods
depending upon the selected chelator. The peptide can generally be most
conveniently
prepared by techniques generally established and known in the art of peptide
synthesis, such
as the solid-phase peptide synthesis (SPPS) approach. Solid-phase peptide
synthesis (SPPS)
involves the stepwise addition of amino acid residues to a growing peptide
chain that is
linked to an insoluble support or matrix, such as polystyrene. The C-terminal
residue of the
peptide is first anchored to a commercially available support with its amino
group protected
with an N-protecting agent such as a t-butyloxycarbonyI group (Boc) or a
fluorenylmethoxycarbonyl (Fmoc) group. The amino protecting group is removed
with
suitable deprotecting agents such as TFA in the case of Boc or piperidine for
Fmoc and the
next amino acid residue (in N-protected form) is added with a coupling agent
such as N,N'-
dicyclohexylcarbodiimide (DCC), orN,N'-diisopropylcarbodiimide (DIC) or 2-(1H-
benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU). Upon
formation of a peptide bond, the reagents are washed from the support. After
addition of the
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final residue, the peptide is cleaved from the support with a suitable reagent
such as
trifluoroacetic acid (TFA) or hydrogen fluoride (HF).
[00181] The linker may then be coupled to form a conjugate by reacting the
free amino
group of the Trp$ residue of the GRP receptor targeting peptide with an
appropriate
functional group ofthe linker. The entire construct of chelator, linker and
targeting moiety
discussed above may also be assembled on resin and then cleaved by agency of
suitable
reagents such as trifluoroacetic acid or HF, as well.
[00182] Bombesin (7-14) is subject to proteolytic cleavage in vitro and in
vivo, which
shortens the half life of the peptide. It is well known in the literature that
the amide bond of
10 the backbone of the polypeptide may be substituted and retain activity,
while resisting
proteolytic cleavage. For example, to reduce or eliminate undesired
proteolysis, or other
degradation pathways that diminish serum stability, resulting in reduced or
abolished
bioactivity, or to restrict or increase conformational flexibility, it is
common to substitute
amide bonds within the backbone of the peptides with functionality that mimics
the existing
15 conformation or alters the conformation in the manner desired. Such
modifications may
produce increased binding affinity or improved pharmacokinetic behavior. It is
understood
that those knowledgeable in the art of peptide synthesis can make the
following amide bond-
changes for any amide bond connecting two amino acids (e.g., amide bonds in
the targeting
moiety, linker, chelator, etc.) with the expectation that the resulting
peptides could have the
20 same or improved activity: insertion of alpha-N-methylamides or backbone
thioamides,
removal of the carbonyl to produce the cognate secondary amines, replacement
of one amino
acid with an aza-aminoacid to produce semicarbazone derivatives, and use of E-
olefins and
substituted E-olefins as amide bond surrogates. The hydrolysis can also be
prevented by
incorporation of a D-amino acid of one of the amino acids of the labile amide
bond, or by
25 alpha-methyl aminoacid derivatives. Backbone amide bonds have also been
replaced by
heterocycles such as oxazoles, pyrrolidinones, imidazoles, as well as
ketomethylenes and
fluoroolefins.
[00183] Some specific compounds including such amide bond modifications are
listed in
Table 4a. The abbreviations used in Table 4a for the various amide bond
modifications are
30 exemplified below:
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51
H2N O H2N O
~ H~ H O
W N N W Ni N
H p II
O
~ NH ~ NH
Q W
NMeQ W \
H2N O H2N O
H O H
~N~ N~ ~N~ _''J~~.~',N
H S H
NH ~ NH
Q 'I~ [CSNH] W
Q ~ [CH2NH) W \ /
H2N O H2N O
O ... H
WN / _ ~N N
H H O
NH ~ NH
Q'Y [CH=CH] W \ ~' a.-MeQ W \ /
TABLE 4A
Table
4A
-
Preferred
Amide
Bond
Modified
Analogs
_
Comp M_N-O-P BBN
Analogue
ound
D03A-
_
monoamide-G-~'neW A V G H L M-NHZ
~Oi Abz4
D03A- Q-
L402 monoamide-G-'Y[CSW A V G H L M-NHZ
Abz4 NH
D03A- Q-
L403 monoamide-G-'Y[CHW A V G H L M-NHZ
Abz4 ZNH
D03A- Q-
L404 monoamide-G-'Y[CHW A V G H L M-NHZ
Abz4 =CH
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52
Table
4A
-
Preferred
Amide
Bond
Modified
Analo
s
Comp M-N-O-P BBN
Analogue
ound
D03A-
monoamide-G-Me W A V G H L M-NHz
~06 Abz4 Q
DO3A-
monoamide-G-Q Nme-W A V G H L M-NHz
L4D6 Abz4
D03A-
monoamide-G-Q ~ A V G H L M-NHZ
L407 CSC]
Abz4
D03A-
monoamide-G-Q A V G H L M-NHz
L408 iyCCHZNH]
Abz4 - - -,
D03A-
monoamide-G-Q A V G H L M-NHz
L4D9 (CH=CH]
Abz4
DO3A-
monoamide-G-Q a -MeW A V G H L M-NHz
L410 Abz4 .-.
DO3A-
monoamide-G-Q W Nme-A V G H L M-NHz
L411 Abz4
DO3A- A-
412 monoamide-G-Q W ,1,[CSC]V G H L M-NHz
L Abz4
D03A- A_
monoamide-G-Q W V G H L M-NHz
L413 ~,[CHZ~]
Abz4
D03A-
monoamide-G-Q W Aib V G H L M-NHz
L414 Abz4
D03A-
mnoamide-G-Q W A V Sar H L M-NHz
L415 Abz4
D03A-
monoamide-G-Q W A V ~~CS~] H L M-NHz
L416 Abz4
D03A- _
G
monoamide-G-Q W A V H L M-NHz
'Y[CH=CH]
L417 Abz4
D03A-
monoamide-G-Q W A V Dala H L M-NHz
L418 Abz4
D03A-
monoamide-G-Q W A V G Nme-His L M-NHZ
L419 Abz4
D03A- g_
monoamide-G-Q W A V G ~,[CSC] L M-NHz
L420 Abz4
D03A- H_
monoamide-G-Q W A V G ~,[CH L M-NHz
~]
L,421Abz4 2
D03A- H-
monoamide-G-Q W A V ~ G ~ ~y[CH=CH]'L [ M
~z
Lt122Abz4
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WO 2005/067983 PCT/US2004/022115
53
_
Table
4A
-
Preferred
Amide
Bond
Modified
Analo
s
Comp M_N-O-P BBN
Analogue
ound
D03A-
L423 monoamide-G-Q W A V G a. -MeH L M-NHZ
Abz4
D03A-
monoamide-G-Q W A V G H Lme- M-NHZ
L424 Abz4
D03A-
L425 'nonoamide-G-Q W A V G H M NHZ
Abz4 MeL
D03A-
L300 monoamide-G-Q W A V G H F-L NHZ
ABz4
4. Labeling And Administration Of Radiopharmaceutical Compounds
[00184] Incorporation of the metal within the radiopharmaceutical conjugates
can be
achieved by various methods commonly known in the art of coordination
chemistry. When
the metal is 99mTC, a preferred radionuclide for diagnostic imaging, the
following general
procedure can be used to form a technetium complex. A peptide-chelator
conjugate solution
is formed by initially dissolving the conjugate in water, dilute acid, or in
an aqueous solution
of an alcohol such as ethanol. The solution is then optionally degassed to
remove dissolved
oxygen. When an -SH group is present in the peptide, a thiol protecting group
such as Acm
(acetamidomethyl), trityI or other thiol protecting group may optionally be
used to protect the
thiol from oxidation. The thiol protecting groups) are removed with a suitable
reagent, for
example with sodium hydroxide, and are then neutralized with an organic acid
such as acetic
acid (pH 6.0-6.5). Alternatively, the thiol protecting group can be removed in
situ during
I S technetium chelation. In the labeling step, sodium pertechnetate obtained
from a
molybdenum generator is added to a solution of the conjugate with a sufficient
amount of a
reducing agent, such as stannous chloride, to reduce technetium and is either
allowed to stand
at room temperature or is heated. The labeled conjugate can be separated from
the
contaminants 99mTc04 and colloidal 99mTc02 chromatographically, for example
with a C-18
Sep Pak cartridge [Millipore Corporation, Waters Chromatography Division, 34
Maple
Street, Milford, Massachusetts 01757] or by HPLC using methods known to those
skilled in
the art.
[00185] In an alternative method, the labeling can be accomplished by a
transchelation
reaction. In this method, the technetium source is a solution of technetium
that is reduced
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54
and complexed with labile ligands prior to reaction with the selected
chelator, thus facilitating
ligand exchange with the selected chelator. Examples of suitable ligands for
transchelation
includes tartrate, citrate, gluconate, and heptagluconate. It will be
appreciated that the
conjugate can be labeled using the techniques described above, or
alternatively, the chelator
itself may be labeled and subsequently coupled to the peptide to form the
conjugate; a
process referred to as the "prelabeled chelate" method. Re and Tc are both in
row VIIB of
the Periodic Table and they are chemical congeners. Thus, for the most part,
the
complexation chemistry of these two metals with ligand frameworks that exhibit
high in vitro
and in vivo stabilities are the same [Eckelman, 1995] and similar chelators
and procedures
can be used to label with Re. Many 99"'Tc or lg6»gBRe complexes, which are
employed to
form stable radiometal complexes with peptides and proteins, chelate these
metals in their +5
oxidation state [Lister-James et al., 1997). This oxidation state makes it
possible to
selectively place 99"'Tc- or ~86~~88Re into ligand frameworks already
conjugated to the
biomolecule, constructed from a variety of 99mTc(V) and/or 186~~88Re(V) weak
chelates (e.g.,
99"'Tc- glucoheptonate, citrate, gluconate, etc.) [Eckelman, 1995; Lister-
James et al., 1997;
Pollak et al., 1996]. These references are hereby incorporated by reference in
their entirety.
5. Diagnostic and Therapeutic Uses
[00186) When labeled with diagnostically and/or therapeutically useful metals
or optical
labels, compounds of the present invention can be used to treat and/or detect
any pathology
involving overexpression of GRP receptors (or NMB receptors) by procedures
established in
the art of radiodiagnostics, radiotherapeutics and optical imaging. [See,
e.g., Bushbaum,
1995; Fischman et al., 1993; Schubiger et al., 1996; Lowbertz et al., 1994;
I~renning et al.,
1994; examples of optical dyes include, but are not limited to those described
in WO
98/18497, WO 98/18496, WO 98/18495, WO 98/18498, WO 98/53857, WO 96/17628, WO
97/18841, WO 96/23524, WO 98/47538, and references cited therein, hereby
incorporated by
reference in their entirety.]
[00187] GRP-R expression is highly upregulated in a variety of human tumors.
See e.g.,
WO 99/62563. Thus, compounds of the invention may be widely useful in treating
and
diagnosing cancers, including prostate cancer (primary and metastatic), breast
cancer
(primary and metastatic), colon cancer, gastric cancer, pancreatic cancer, non
small cell lung
cancer, small cell lung cancer, gastrinomas, melanomas, glioblastomas,
neuroblastomas,
uterus leiomyosarcoma tumors, prostatic intraepithelial neoplasias [PIN], and
ovarian cancer.
Additionally, compounds of the invention may be useful to distinguish between
conditions in
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WO 2005/067983 PCT/US2004/022115
which GRP receptors are upregulated and those in which they are not (e.g.
chronic
pancreatitis and ductal pancreatic carcinoma, respectively
[00188] The compounds of the invention, which, as explained in more detail in
the
Examples, show greater specificity and higher uptake in tumors in vivo than
compounds
5 without the novel linkers disclosed herein, exhibit an improved ability to
target GRP
receptor-expressing tumors and thus to image or deliver radiotherapy to these
tissues.
Indeed, as shown in the Examples, radiotherapy is more effective (and survival
time
increased) using compounds of the invention.
[00189] The diagnostic application of these compounds can be as a first line
diagnostic
10 screen for the presence of neoplastic cells using scintigraphic, optical,
sonoluminescence or
photoacoustic imaging, as an agent for targeting neoplastic tissue using hand-
held radiation
detection instrumentation in the field of radioimmuno guided surgery (RIGS),
as a means to
obtain dosimetry data prior to administration of the matched pair
radiotherapeutic compound,
and as a means to assess GRP receptor population as a function of treatment
over time.
15 [00190] The therapeutic application of these compounds can be defined as an
agent that
will be used as a first line therapy in the treatment of cancer, as
combination therapy where
these agents could be utilized in conjunction with adjuvant chemotherapy,
and/or as a
matched pair therapeutic agent. The matched pair concept refers to a single
unmetallated
compound which can serve as both a diagnostic and a therapeutic agent
depending on the
20 radiometal that has been selected for binding to the appropriate chelate.
If the chelator cannot
accommodate the desired metals, appropriate substitutions can be made to
accommodate the
different metal while maintaining the pharmacology such that the behavior of
the diagnostic
compound in vivo can be used to predict the behavior of the radiotherapeutic
compound.
When utilized in conjunction with adjuvant chemotherapy any suitable
chemotherapeutic
25 may be used, including for example, antineoplastic agents, such as platinum
compounds (e.g.,
spiroplatin, cisplatin, and carboplatin), methotrexate, adriamycin, mitomycin,
ansamitocin,
bleomycin, cytosine, arabinoside, arabinosyl adenine, mercaptopolylysine,
vincristine,
busulfan, chlorambucil, melphalan (e.g., PAM, a, L -PAM or phennylalanine
mustard),
mercaptopurine, mitotane. procarbazine hydrochloride, dactinomycin
(actinomycin D),
30 daunorubcin hydrochloride, doxorubicin hydrochloride, taxol, mitomycin,
plicamycin
(mithramycin), aminoglutethimide, estramustine phosphate sodium, flutamide,
leuprolide
acetate, megestrol acetate, tamoxifen citrate, testolactone, trilostane,
arnsacrine (m-AMSA),
asparaginase (L-asparaginase) EYwina aparaginase, etoposide -(VP-16),
interferon a,-2a,
CA 02549318 2006-06-08
WO 2005/067983 PCT/US2004/022115
56
interferon a-2b, teniposide (VM-26), vinblastine sulfate (VLB), and
arabinosyl. In certain
embodiments, the therapeutic may be monoclonal antibody, such as a monoclonal
antibody
capable of binding to melanoma antigen.
[00191] A conjugate labeled with a radionuclide metal, such as 99mTc, can be
administered
to a mammal, including human patients or subjects, by, for example,
intravenous,
subcutaneous or intraperitoneal injection in a pharmaceutically acceptable
carrier and/or
solution such as salt solutions like isotonic saline. Radiolabeled
scintigraphic imaging agents
provided by the present invention are provided having a suitable amount of
radioactivity. In
forming 99"'Tc radioactive complexes, it is generally preferred to form
radioactive complexes
in solutions containing radioactivity at concentrations of from about 0.01
millicurie (mCi) to
100 mCi per mL. Generally, the unit dose to be administered has a
radioactivity of about
0.01 mCi to about 100 mCi, preferably 1 mCi to 30 mCi. The solution to be
injected at unit
dosage is from about 0.01 mL to about 10 mL. The amount of labeled conjugate
appropriate
for administration is dependent upon the distribution profile of the chosen
conjugate in the
sense that a rapidly cleared conjugate may need to be administered in higher
doses than one
that clears less rapidly. In vivo distribution and localization can be tracked
by standard
scintigraphic techniques at an appropriate time subsequent to administration;
typically
between thirty minutes and 180 minutes depending upon the rate of accumulation
at the target
site with respect to the rate of clearance at non-target tissue. For example,
after injection of
the diagnostic radionuclide-labeled compounds of the invention into the
patient, a gamma
camera calibrated for the gamma ray energy of the nuclide incorporated in the
imaging agent
can be used to image areas of uptake of the agent and quantify the amount of
radioactivity
present in the site. Imaging of the site izz vivo can take place in a few
minutes. However,
imaging can take place, if desired, hours or even longer, after the
radiolabeled peptide is
injected into a patient. In most instances, a sufficient amount of the
administered dose will
accumulate in the area to be imaged within about 0.1 hour to permit the taking
of
scintiphotos.
[00192] The compounds of the present invention can be administered to a
patient alone or
as part of a composition that contains other components such as excipients,
diluents, radical
scavengers, stabilizers, and carriers, all of which are well-known in the art.
The compounds
can be administered to patients either intravenously or intraperitoneally.
[00193] There are numerous advantages associated with the present invention.
The
compounds made in accordance with the present invention form stable, well-
defined 99mTc or
CA 02549318 2006-06-08
WO 2005/067983 PCT/US2004/022115
57
~ssnssRe labeled compounds. Similar compounds of the invention can also be
made by using
appropriate chelator frameworks for the respective radiometals, to form
stable, well-defined
products labeled with ' S3Sm, g°Y, ' 66I-Io, ~os~~ 199Au~ la9Pm, '
~~Lu, "'In or other radiometals.
The radiolabeled GRP receptor targeting peptides selectively bind to
neoplastic cells
expressing GRP receptors, and if an agonist is used, become internalized, and
are retained in
the tumor cells for extended time periods. The radioactive material that does
not reach (i.e.,
does not bind) the cancer cells is preferentially excreted efficiently into
the urine with
minimal retention of the radiometal in the kidneys.
6. Optical Imaginn~, Sonoluminescence, Photoacoustic Imaging and
Phototherapy
[00194] In accordance with the present invention, a number of optical
parameters may be
employed to determine the location of a target with in vivo light imaging
after injection of the
subject with an optically-labeled compound of the invention. Optical
parameters to be
detected in the preparation of an image may include transmitted radiation,
absorption,
fluorescent or phosphorescent emission, light reflection, changes in
absorbance amplitude or
maxima, and elastically scattered radiation. For example, biological tissue is
relatively
translucent to light in the near infrared (NIR) wavelength range of 650-1000
nm. NIR
radiation can penetrate tissue up to several centimeters, permitting the use
of compounds of
the present invention to image target-containing tissue in vivo. The use of
visible and near-
infrared (NIR) light in clinical practice is growing rapidly. Compounds
absorbing or emitting
in the visible, NIR, or long-wavelength (UV-A, >350 nm) region of the
electromagnetic
spectrum are potentially useful for optical tomographic imaging, endoscopic
visualization,
and phototherapy.
[00195] A major advantage of biomedical optics lies in its therapeutic
potential.
Phototherapy has been demonstrated to be a safe and effective procedure for
the treatment of
various surface lesions, both external and internal. Dyes are important to
enhance signal
detection and/or photosensitizing of tissues in optical imaging and
phototherapy. Previous
studies have shown that certain dyes can localize in tumors and serve as a
powerful probe for
the detection and treatment of small cancers (D. A. Bellnier et al., Murine
pharmacokinetics
and antitumor efficacy of the photodynamic sensitizer 2-[1-hexyloxyethyl]-2-
devinyl
pyropheophorbide-a, J. Photochem. Photobiol., 1993, 20, pp. 55-61; G. A.
Wagnieres et al.,
In vivo fluorescence spectroscopy and imaging for oncological applications,
Photochem.
Photobiol., 1998, 68, pp. 603-632; J. S. Reynolds et al., Imaging of
spontaneous canine
CA 02549318 2006-06-08
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58
mammary tumors using fluorescent contrast agents, Photochem. Photobiol., 1999,
70, pp. 87-
94). All of these listed references are hereby incorporated by reference in
their entirety.
However, these dyes do not localize preferentially in malignant tissues.
[00196] In an exemplary embodiment, the compounds of the invention may be
conjugated
with photolabels, such as optical dyes, including organic chromophores or
fluorophores,
having extensive delocalized ring systems and having absorption or emission
maxima in the
range of 400-1500 nm. The compounds of the invention may alternatively be
derivatized
with a bioluminescent molecule. The preferred range of absorption maxima for
photolabels
is between 600 and 1000 mn to minimize interference with the signal from
hemoglobin.
Preferably, photoabsorption labels have large molar absorptivities, e.g., >
105 cm-~M-~, while
fluorescent optical dyes will have high quantum yields. Examples of optical
dyes include,
but are not limited to those described in US 6,641,798, WO 98/18497, WO
98/18496, WO
98/18495, WO 98118498, WO 98/53857, WO 96/17628, WO 97/18841, WO 96/23524, WO
98/47538, and references cited therein, all hereby incorporated by reference
in their entirety.
For example, the photolabels may be covalently linked directly to compounds of
the
invention, such as, for example, compounds comprised of GRP receptor targeting
peptides
and linkers of the invention. Several dyes that absorb and emit light in the
visible and near-
infrared region of electromagnetic spectrum are currently being used for
various biomedical
applications due to their biocompatibility, high molar absorptivity, and/or
high fluorescence
quantum yields. The high sensitivity of the optical modality in conjunction
with dyes as
contrast agents parallels that of nuclear medicine, and permits visualization
of organs and
tissues without the undesirable effect of ionizing radiation. Cyanine dyes
with intense
absorption and emission in the near-infrared (NIR) region are particularly
useful because
biological tissues are optically transparent in this region (B. C. Wilson,
Optical properties of
tissues. Encyclopedia of Human Biology, 1991, 5, 587-597). For example,
indocyanine
green, which absorbs and emits in the NIR region has been used for monitoring
cardiac
output, hepatic functions, and liver blood flow (Y-L. He, H. Tanigami, H.
Ueyama, T.
Mashimo, and I. Yoshiya, Measurement of blood volume using indocyanine green
measured
with pulse-spectrometry: Its reproducibility and reliability. Critical Care
Medicine, 1998,
26(8), 1446-1451; J. Caesar, S. Shaldon, L. Chiandussi, et al., The use of
Indocyanine green
in the measurement of hepatic blood flow and as a test of hepatic function.
Clin. Sci. 1961,
21, 43-57) and its functionalized derivatives have been used to conjugate
biomolecules for
diagnostic purposes (R. B. Mujumdar, L. A. Ernst, S. R. Mujumdar, et al.,
Cyanine dye
CA 02549318 2006-06-08
WO 2005/067983 PCT/US2004/022115
59
labeling reagents: Sulfoindocyanine succinimidyl esters. Bioconjugate
Chemistry, l 993, 4(2),
105-111; Linda G. Lee and Sam L. Woo. "N-Heteroaromatic ion and iminium ion
substituted
cyanine dyes for use as fluorescent labels", U.S. Pat. No. 5,453,505; Eric
Hohenschuh, et al.
"Light imaging contrast agents", WO 98/48846; Jonathan Turner, et al. "Optical
diagnostic
agents for the diagnosis of neurodegenerative diseases by means of near infra-
red radiation",
WO 98/22146; Kai Licha, et al. "In-vivo diagnostic process by near infrared
radiation", WO
96/17628; Robert A. Snow, et al., Compounds, WO 98/48838, US 6,641,798. All of
these
listed references are hereby incorporated by reference in their entirety.
[00197] After injection of the optically-labeled compound, the patient is
scanned with one
or more light sources (e.g., a laser) in the wavelength range appropriate for
the photolabel
employed in the agent. The light used may be monochromatic or polychromatic
and
continuous or pulsed. Transmitted, scattered, or reflected light is detected
via a photodetector
tuned to one or multiple wavelengths to determine the location of target-
containing tissue
(e.g., tissue containing GRP) in the subject. Changes in the optical parameter
may be
monitored over time to detect accumulation of the optically-labeled reagent at
the target site
(e.g., the tumor or other site with GRP receptors). Standard image processing
and detecting
devices may be used in conjunction with the optical imaging reagents of the
present
invention.
[00198] The optical imaging reagents described above may also be used for
acousto-
optical or sonoluminescent imaging performed with optically-labeled imaging
agents (see
U.S. 5,171,298, WO 98/57666, and references therein). In acousto-optical
imaging,
ultrasound radiation is applied to the subject and affects the optical
parameters of the
transmitted, emitted, or reflected light. In sonoluminescent imaging, the
applied ultrasound
actually generates the light detected. Suitable imaging methods using such
techniques are
described in WO 98/57666.
[00199] Various imaging techniques and reagents are described in U.S. Patents
6,663,847,
6,656,451, 6,641,798, 6,485,704, 6,423,547, 6,395,257, 6,280,703, 6,277,841,
6,264,920,
6,264,919, 6,228,344, 6,217,848, 6,190,641, 6,183,726, 6,180,087, 6,180,086,
6,180,085,
6,013,243, and published U.S. Patent Applications 2003185756, 20031656432,
2003158127,
2003152577, 2003143159, 2003105300, 2003105299, 2003072763, 2003036538,
2003031627, 2003017164, 2002169107, 2002164287, and 2002156117, all of which
are
hereby incorporated by reference.
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7. Radiotherapy
[00200] Radioisotope therapy involves the administration of a radiolabeled
compound in
sufficient quantity to damage or destroy the targeted tissue. After
administration of the
compound (by e.g., intravenous, subcutaneous, or intraperitonal injection),
the radiolabeled
5 pharmaceutical localizes preferentially at the disease site (in this
instance, tumor tissue or
other tissue that expresses the pertinent GRP receptor). Once localized, the
radiolabeled
compound then damages or destroys the diseased tissue with the energy that is
released
during the radioactive decay of the isotope that is administered. As discussed
herein, the
invention also encompasses use of radiotherapy in combination with adjuvant
chemotherapy
10 (or in combination with any other appropriate therapeutic agent).
[00201 ] The design of a successful radiotherapeutic involves several critical
factors:
1. selection of an appropriate targeting group to deliver the radioactivity
to the disease site;
2. selection of an appropriate radionuclide that releases sufficient energy
15 to damage that disease site, without substantially damaging adjacent normal
tissues; and
3. selection of an appropriate combination of the targeting group and the
radionuclide without adversely affecting the ability of this conjugate to
localize at the disease
site. For radiometals, this often involves a chelating group that coordinates
tightly to the
radionuclide, combined with a linker that couples said chelate to the
targeting group, and that
20 affects the overall biodistribution of the compound to maximize uptake in
target tissues and
minimize uptake in normal, non-target organs.
[00202] The present invention provides radiotherapeutic agents that satisfy
all three of
the above criteria, through proper selection of targeting group, radionuclide,
metal chelate
and linker.
25 [00203] Radiotherapeutic agents may contain a chelated 3+ metal ion from
the class of
elements known as the lanthanides (elements of atomic number 57-71) and their
analogs (i.e.
M3+ metals such as yttrium and indium). Typical radioactive metals in this
class include the
isotopes 90-Yttrium, 111-Indium, 149-Promethium, 153-Samarium, 166-Dysprosium,
166-
Holmium, 175-Ytterbium, and 1~~-Lutetium. All of these metals (and others in
the lanthanide
30 series) have very similar chemistries, in that they remain in the +3
oxidation state, and prefer
to chelate to ligands that bear hard (oxygen/nitrogen) donor atoms, as
typified by derivatives
of the well known chelate DTPA (diethylenetriaminepentaacetic acid) and
polyaza-
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61
polycarboxylate macrocycles such as DOTA (1,4,7,10-tetrazacyclododecane-N,
N',N",N"'-
tetraacetic acid and its close analogs. The structures of these chelating
ligands, in their fully
deprotonated form are shown below.
DTPA DOTA
O _.
O - -OOC----~
,O ,r-~
j ~ COO-
O~ N N
~
N~
O N N
N ~
N~
- ~ ~ ,
-OOC-J
~-/ ~--COO-
O
.
O O_
O
[00204] These chelating ligands encapsulate the radiometal by binding to it
via
multiple nitrogen and oxygen atoms, thus preventing the release of free
(unbound) radiometal
into the body. This is important, as in vivo dissociation of 3~ radiometals
from their chelate
can result in uptake of the radiometal in the liver, bone and spleen
[Brechbiel MW, Gansow
OA, "Backbone-substituted DTPA ligands for 9°Y radioimmunotherapy",
Bioconj. Chem.
1991; 2:187-194; Li, WP, Ma DS, Higginbotham C, Hoffinan T, Ketring AR, Cutler
CS,
Jurisson, SS, "Development of an in vitro model for assessing the in vivo
stability of
lanthanide chelates." Nucl. Med. Biol. 2001; 28(2): 145-154; Kasokat T, Urich
K. Arzneim.-
Forsch, "Quantification of dechelation of gadopentetate dimeglumine in rats".
1992; 42(6):
869-76]. Unless one is specifically targeting these organs, such non-specific
uptake is highly
undesirable, as it leads to non-specific irradiation of non-target tissues,
which can lead to
such problems as hematopoietic suppression due to irradiation of bone marrow.
[00205] For radiotherapy applications any of the chelators for therapeutic
radionuclides disclosed herein may be used. However, forms of the DOTA chelate
[Tweedle
MF, Gaughan GT, Hagan JT, "1-Substituted-1,4,7-triscarboxymethyl-1,4,7,10-
tetraazacyclododecane and analogs." US Patent 4,885,363, Dec. 5, 1989] are
particularly
preferred, as the DOTA chelate is expected to de-chelate less in the body than
DTPA or other
linear chelates. Compounds L64 and L70 (when labeled with an appropriate
therapeutic
radionuclide) are particularly preferred for radiotherapy.
[00206] General methods for coupling DOTA-type macrocycles to targeting groups
through a linker (e.g., by activation of one of the carboxylates of the DOTA
to form an active
CA 02549318 2006-06-08
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62
ester, which is then reacted with an amino group on the linker to form a
stable amide bond),
are known to those skilled in the art. (See, e.g., Tweedle et al. US Patent
4,885,363).
Coupling can also be performed on DOTA-type macrocycles that are modified on
the
backbone of the polyaza ring.
[00207) The selection of a proper nuclide for use in a particular
radiotherapeutic
application depends on many factors, including:
a. Physical half life - This should be long enough to allow synthesis and
purification of the radiotherapeutic construct from radiometal and conjugate,
and delivery of
said construct to the site of injection, without significant radioactive decay
prior to injection.
Preferably, the radionuclide should have a physical half life between about
0.5 and 8 days.
b. Energy of the emissions) from the radionuclide - Radionuclides that
are particle emitters (such as alpha emitters, beta emitters and Auger
electron emitters) are
particularly useful, as they emit highly energetic particles that deposit
their energy over short
distances, thereby producing highly localized damage. Beta emitting
radionuclides are
particularly preferred, as the energy from beta particle emissions from these
isotopes is
deposited within 5 to about 150 cell diameters. Radiotherapeutic agents
prepared from these
nuclides are capable of killing diseased cells that are relatively close to
their site of
localization, but cannot travel long distances to damage adjacent normal
tissue such as bone
marrow.
c. Specific activit~(i.e. radioactivity per mass of the radionuclide~ -
Radionuclides that have high specific activity (e.g., generator produced 90-Y,
111-In, 177-
Lu) are particularly preferred. The specific activity of a radionuclide is
determined by its
method of production, the particular target that is used to produce it, and
the properties of the
isotope in question.
[00208] Many of the lanthanides and lanthanoids include radioisotopes that
have
nuclear properties that make them suitable for use as radiotherapeutic agents,
as they emit
beta particles. Some of these are listed in the table below.
Approximate
range of b-
Half -Life Max b- energy Gamma energy particle
Isoto a da s Me ke cell diameters
i49-Pm 2.21 1.1 286 60
's3-Sm 1.93 0.69 103 30
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63
Approximate
range of b-
Half -Life Max b- energy Gamma energy particle
Isoto a da s MeV ke cell diameters
'6~-Dy 3.40 0.40 82.5 15
166-HO 1.12 1.8 80.6 117
'~5-Yb 4.19 0.47 396 17
'~~-Lu 6.71 0.50 208 20
9o-Y 2.67 2.28 - -150
"'-In 2.810 Auger electron 173, 247 < S~m
emitter
Pm:Promethium, Sm:Samarium, Dy:Dysprosium, Ho:Holmium, Yb:Ytterbium,
Lu:Lutetium, Y:Yttrium, In:Indium
[00209] Methods for the preparation of radiometals such as beta-emitting
lanthanide
radioisotopes are known to those skilled in the art, and have been.described
elsewhere [e.g.,
Cutler C S, Smith CJ, Ehrhardt GJ.; Tyler TT, Jurisson SS, Deutsch E. "Current
and potential
therapeutic uses of lanthanide radioisotopes." Cancer Biother. Radiopharm.
2000; 15(6):
531-545]. Many ofthese isotopes can be produced in high yield for relatively
low cost, and
many (e.g. 9o-Y,'ø9-Pm,'~~_Lu) can be produced at close to Garner-free
specific activities (i.e.
the vast majority of atoms are radioactive). Since non-radioactive atoms can
compete with
their radioactive analogs for binding to receptors on the target tissue, the
use of high specific
activity radioisotope is important, to allow delivery of as high a dose of
radioactivity to the
target tissue as possible.
[00210] Radiotherapeutic derivatives of the invention containing beta-emitting
isotopes of rhenium (' 86-Re and ' $8-Re) are also particularly preferred.
8. Dosages And Additives
[00211] Proper dose schedules for the compounds of the present invention are
known
to those skilled in the art. The compounds can be administered using many
methods which
include, but are not limited to, a single or multiple IV or IP injections. For
radiopharmaceuticals, one administers a quantity of radioactivity that is
sufficient to permit
imaging or, in the case of radiotherapy, to cause damage or ablation of the
targeted GRP-R
bearing tissue, but not so much that substantive damage is caused to non-
target (normal
tissue). The quantity and dose required for scintigraphic imaging is discussed
supra. The
quantity and dose required for radiotherapy is also different for different
constructs,
depending on the energy and half life of the isotope used, the degree of
uptake and clearance
CA 02549318 2006-06-08
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64
of the agent from the body and the mass of the tumor. In general, doses can
range from a
single dose of about 30-50 mCi to a cumulative dose of up to about 3 Curies.
[00212] For optical imaging compounds, dosages sufficient to achieve the
desired
image enhancement are known to those skilled in the art and may vary widely
depending on
the dye or other compound used, the organ or tissue to be imaged, the imaging
equipment
used, etc.
[00213] The compositions of the invention can include physiologically
acceptable
buffers, and can require radiation stabilizers to prevent radiolytic damage to
the compound
prior to injection. Radiation stabilizers are known to those skilled in the
art, and may include,
for example, para-aminobenzoic acid, ascorbic acid, gentistic acid and the
like.
[00214] A single, or mufti-vial kit that contains all of the components needed
to
prepare the diagnostic or therapeutic agents of this invention is an integral
part of this
invention. In the case of radiopharmaceuticals, such kits will often include
all necessary
ingredients except the radionuclide.
[00215] For example, a single-vial kit for preparing a radiopharmaceutical of
the
invention preferably contains a chelator/linker/targeting peptide conjugate of
the formula M-
N-O-P-G, a source of stannous salt (if reduction is required, e.g., when using
technetium), or
other pharmaceutically acceptable reducing agent, and is appropriately
buffered with
pharmaceutically acceptable acid or base to adjust the pH to a value of about
3 to about 9.
The quantity and type of reducing agent used will depend highly on the nature
of the
exchange complex to be formed. The proper conditions are well known to those
that are
skilled in the art. It is preferred that the kit contents be in lyophilized
form. Such a single
vial kit may optionally contain labile or exchange ligands such as
glucoheptonate, gluconate,
mannitol, malate, citric or tartaric acid and can also contain reaction
modifiers such as
diethylenetriamine-pentaacetic acid (DPTA), ethylenediamine tetraacetic acid
(EDTA), or a,
13, or y-cyclodextrin that serve to improve the radiochemical purity and
stability of the final
product. The kit may also contain stabilizers, bulking agents such as
mannitol, that are
designed to aid in the freeze-drying process, and other additives known to
those skilled in the
art.
[00216] A mufti-vial kit preferably contains the same general components but
employs
more than one vial in reconstituting the radiopharmaceutical. For example, one
vial may
contain all of the ingredients that are required to form a labile Tc(V)
complex on addition of
CA 02549318 2006-06-08
WO 2005/067983 PCT/US2004/022115
pertechnetate (e.g. the stannous source or other reducing agent).
Pertechnetate is added to
this vial, and after waiting an appropriate period of time, the contents of
this vial are added to
a second vial that contains the chelator and targeting peptide, as well as
buffers appropriate to
adjust the pH to its optimal value. After a reaction time of about 5 to 60
minutes, the
5 complexes of the present invention are formed. It is advantageous that the
contents of both
vials of this multi-vial kit be lyophilized. As above, reaction modifiers,
exchange ligands,
stabilizers, bulking agents, etc. may be present in either or both vials.
General Preuaration Of Compounds
[00217] The compounds of the present invention can be prepared by various
methods
10 depending upon the selected chelator. The peptide portion of the compound
can be most
conveniently prepared by techniques generally established and known in the art
of peptide
synthesis, such as the solid-phase peptide synthesis (SPPS) approach. Because
it is amenable
to solid phase synthesis, employing alternating FMOC protection and
deprotection is the
preferred method of making short peptides. Recombinant DNA technology is
preferred for
15 producing proteins and long fragments thereof.
[00218] Solid-phase peptide synthesis (SPPS) involves the stepwise addition of
amino
acid residues to a growing peptide chain that is linked to an insoluble
support or matrix, such
as polystyrene. The C-terminal residue of the peptide is first anchored to a
commercially
available support with its amino group protected with an N-protecting agent
such as a t-
20 butyloxycarbonyl group (Boc) or a fluorenylmethoxycarbonyl (Fmoc) group.
The amino
protecting group is removed with suitable deprotecting agents such as TFA in
the case of Boc
or piperidine for Fmoc and the next amino acid residue (in N-protected form)
is added with a
coupling agent such as diisopropylcarbodiimide (DIC). Upon formation of a
peptide bond, v
the reagents are washed from the support. After addition of the final residue,
the peptide is
25 cleaved from the support with a suitable reagent such as trifluoroacetic
acid (TFA) or
hydrogen fluoride (HF).
Alternative Preparation of the Compounds via Segment Coupling
[00219] The compounds of the invention may also be prepared by the process
known
in the art as segment coupling or fragment condensation (Barlos, K. and Gatos,
D.; 2002
30 "Convergent Peptide Synthesis" in Fmoc Solid Phase Synthesis -A Practical
Approach; Eds.
Chan, W.C. and White, P.D.; Oxford University Press, New York; Chap. 9, pp.
215-228). In
this method segments of the peptide usually in side-chain protected form, are
prepared
CA 02549318 2006-06-08
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66
separately by either solution phase synthesis or solid phase synthesis or a
combination of the
two methods. The choice of segments is crucial and is made using a division
strategy that
can provide a manageable number of segments whose C-terminal residues and N-
terminal
residues are projected to provide the cleanest coupling in peptide synthesis.
The C-terminal
residues of the best segments are either devoid of chiral alpha carbons
(glycine or other
moieties achiral at the carbon oc to the carboxyl group to be activated in the
coupling step) or
are compromised of amino acids whose propensity to racemization during
activation and
coupling is lowest of the possible choices. The choice of N-terminal amino
acid for each
segment is based on the ease of coupling of an activated acyl intermediate to
the amino
group. Once the division strategy is selected the method of coupling of each
of the segments
is chosen based on the synthetic accessibility of the required intermediates
and the relative
ease of manipulation and purification of the resulting products (if needed).
The segments are
then coupled together, both in solution, or one on solid phase and the other
in solution to
prepare the final structure in fully or partially protected form.
[00220] The protected target compound is then subjected to removal of
protecting
groups, purified and isolated o give the final desired compound. Advantages of
the segment
coupling approach are that each segment can be purified separately, allowing
the removal of
side products such as deletion sequences resulting from incomplete couplings
or those
derived from reactions such as side-chain amide dehydration during coupling
steps, or
internal cyclization of side-chains (such as that of Gln) to the alpha amino
group during
deprotection of Fmoc groups. Such side products would all be present in the
final product of
a conventional resin-based 'straight through' peptide chain assembly whereas
removal of
these materials can be performed, if needed, at many stages in a segment
coupling strategy.
Another important advantage of the segment coupling strategy is that different
solvents,
reagents and conditions can be applied to optimize the synthesis of each of
the segments to
high purity and yield resulting in improved purity and yield of the final
product. Other
advantages realized are decreased consumption of reagents and lower costs.
EXAMPLES
[00221] The following examples are provided as examples of different methods
which
can be used to prepare various compounds of the present invention. Within each
example,
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67
there are compounds identified in single bold capital letter (e.g., A, B, C),
which correlate to
the same labeled corresponding compounds in the drawings identified.
General Experimental
A. Definitions of Additional Abbreviations Used
[00222] The following common abbreviations are used throughout this
specification:
l,l-dimethylethoxycarbonyl (Boc or Boc);
9-fluorenylmethyloxycarbonyl (Fmoc);
allyloxycarbonyl (Aloc);
1-hydroxybenozotriazole (HOBt or HOBT);
N,N'-diisopropylcarbodiimide (DIC);
N-methylpyrrolidinone (NMP);
acetic anhydride (Ac20);
(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (iv-Dde);
trifluoroacetic acid (TFA);
Reagent B (TFA:H20:phenolariisopropylsilane, 88:5:5:2);
diisopropylethylamine (DIEA);
O-( 1 H-benzotri azole-I -yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HBTU);
O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniurn
hexafluorphosphate (HATU);
N-hydroxysuccinimide (NHS);
solid phase peptide synthesis (SPPS);
dimethylsulfoxide (DMSO);
dichloromethane (DCM);
dimethylformamide (DMF);
dimethylacetamide (DMA);
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68
1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid (DOTA);
Triisopropylsilane (TIPS);
1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid (DOTA)
( 1 R)-1-[ 1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl]
ethane-1,2-dicarboxylic acid (CMDOTA);
fetal bovine serum (FBS);
human serum albumin (HSA);
human prostate cancer cell line (PC3);
isobutylchloroformate (IBCF);
tributyl amine (TBA);
radiochemical purity (RCP); and
high performance liquid chromatography (HPLC).
B. Materials
[00223] The Fmoc-protected amino acids used were purchased from Nova-Biochem
(San Diego, CA, USA), Advanced Chem Tech (Louisville, KY., USA), Chem-Impex
International (Wood Dale Ill., USA), and Multiple Peptide Systems (San Diego,
CA., USA).
Other chemicals, reagents and adsorbents required for the syntheses were
procured from
Aldrich Chemical Co. (Milwaukee, WI, USA) and VWR Scientific Products
(Bridgeport,
NJ., USA). Solvents for peptide synthesis were obtained from Pharmco Co.
(Brookfield CT.,
USA). Columns for HPLC analysis and purification were obtained from Waters Co.
(Milford, MA., USA). Experimental details are given below for those that were
not
commercially available.
C. Instrumentation for Peptide Synthesis
[00224] Peptides were prepared using an Advanced ChemTech 496 SZ MOS
synthesizer, an Advanced ChemTech 357 FBS synthesizer and/or by manual peptide
synthesis. However the protocols for iterative deprotection and chain
extension employed
were the same for all.
D. Automated synthesis with the Symphony instrument (made by Rainin)
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69
[00225] The synthesis was run with Symphony Software (Version 3) supplied by
Protein Technologies Inc. Novagel TGR resin, with a substitution of 0.25
mmol/g, was used,
and each well contained 0.2 g of the resin (50 ~.mol). The amino acids were
dissolved in
NMP and the concentration was 0.25M. A 0.25M solution of HBTU and N-
Methylmorpholine in DMF was prepared and used for the coupling. All the
couplings were
carried out for 2.0 h. The cleavage was done outside the machine by
transferring the resin to
another reaction vessel and using Reagent B as in the manual synthesis
E. Instrumentation Employed for Analysis and Purification
[00226] Analytical HPLC was performed using a Shimadzu-LC-l0A dual pump
gradient analytical LC system employing Shimadzu-ClassVP software version 4.1
for system
control, data acquisition, and post run processing. Mass spectra were acquired
on a Hewlett-
Packard Series 1100 MSD mass spectrometer interfaced with a Hewlett-Packard
Series 1100
dual pump gradient HPLC system fitted with an Agilent Technologies 1100 series
autosampler fitted for either direct flow injection or injection onto a Waters
Associates
XTerra MS C18 column (4.6 mm x 50 mm, 5~ particle, 120 pore). The instrument
was
driven by a HP Kayak workstation using 'MSD Anyone' software for sample
submission and
HP Chemstation software for instrument control and data acquisition. In most
cases the
samples were introduced via direct injection using a 5 ~L injection of sample
solution at a
concentration of 1 mg/mL and analyzed using positive ion electrospray to
obtain m/e and m/z
(multiply charged) ions for confirmation of structure. 1H-NMR spectra were
obtained on a
Varian Innova spectrometer at 500 MHz. 13C-NMR spectra were obtained on the
same
instrument at 125.73 MHz. Generally the residual 1H absorption, or in the case
of 13C-NMR,
the'3C absorption ofthe solvent employed, was used as an internal reference;
in other cases
tetramethylsilane (8 = 0.00 ppm) was employed. Resonance values are given in 8
units.
Micro analysis data was obtained from Quantitative Technologies Inc.,
Whitehouse NJ.
Preparative HPLC was performed on a Shimadzu-LC-8A dual pump gradient
preparative
HPLC system employing Shimadzu-ClassVP software version 4.3 for system
control, data
acquisition, fraction collection and post run processing.
F. General Procedures for Peptide Synthesis
[00227) Rink Amide-Novagel HL resin (0.6 mmol/g) was used as the solid
support.
G. Coupling Procedure
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[00228] In a typical experiment, the first amino acid was loaded onto 0.1 g of
the resin
(0.06 mmol). The appropriate Fmoc-amino acid in NMP (0.25M solution; 0.960 mL
was
added to the resin followed by N-hydroxybenzotriazole (0.5M in NMP; 0.48 mL)
and the
reaction block (in the case of automated peptide synthesis) or individual
reaction vessel (in
5 the case of manual peptide synthesis) was shaken for about 2 min. To the
above mixture,
diisopropylcarbodiimide (0.5M in NMP; 0.48 mL) was added and the reaction
mixture was
shaken for 4h at ambient temperature. Then the reaction block or the
individual reaction
vessel was purged of reactants by application of a positive pressure of dry
nitrogen.
H. Washing Procedure
10 [00229] Each well of the reaction block was filled with 1.2 mL of NMP and
the block
was shaken for 5 min. The solution was drained under positive pressure of
nitrogen. This
procedure was repeated three times. The same procedure was used, with an
appropriate
volume of NMP, in the case of manual synthesis using individual vessels.
I. Removal of Fmoc Protectin _ Group
15 [00230] The resin bearing the Fmoc-protected amino acid was treated with
1.5 mL of
20% piperidine in DMF (v/v) and the reaction block or individual manual
synthesis vessel
was shaken for 15 min. The solution was drained from the resin. This procedure
was
repeated once and the resin was washed employing the washing procedure
described above.
J. Final coupling of ligand (DOTA and CMDOTA)
20 [00231] The N-terminal amino group of the resin bound peptide linker
construct was
deblocked and the resin was washed. A 0.25M solution of the desired ligand and
HBTU in
NMP was made, and was treated with a two-fold equivalency of DIEA. The
resulting
solution of activated ligand was added to the the resin ( 1.972 mL; 0.48 mmol)
and the
reaction mixture was shaken at ambient temperature for 24-30 h. The solution
was drained
25 and the resin was washed. The final wash of the resin was conducted with
1.5 mL
dichloromethane (3X).
K. Deprotection and purification of the final peptide
[00232] A solution of Reagent B (2 mL; 88:5:5:2 - TFA:phenol:water:TIPS) was
added to the resin and the reaction block or individual vessel was shaken for
4.5h at ambient
30 temperature. The resulting solution containing the deprotected peptide was
drained into a
vial. This procedure was repeated two more times with 1 mL of Reagent B. The
combined
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71
filtrate was concentrated under reduced pressure using a Genevac HT-12 series
II centrifugal
concentrator. The residue in each vial was then triturated with 2 mL of Et20
and the
supernatant was decanted. This procedure was repeated twice to provide the
peptides as
colorless solids. The crude peptides were dissolved in water/acetonitrile and
purified using
either a Waters XTerra MS C18 preparative HPLC column (50 mm x 19 mm, 5 micron
particle size, 120 pore size) or a Waters-YMC C18 ODS column (250 mm x 30 mm
i.d., 10
micron particle size. 120 ~ pore size). The product-containing fractions were
collected and
analyzed by HPLC. The fractions with >95% purity were pooled and the peptides
isolated by
lyophilization.
[00233] Conditions for Preparative HPLC (Waters XTerra Column):
Elution rate: 50 mL/min
Detection: UV, ~, = 220 nm
Eluent A: 0.1 % aq. TFA; Eluent B: Acetonitrile (0.1 % TFA).
Conditions for HPLC Analysis:
Column: Waters XTerra (Waters Co..; 4.6 x 50 mm; MS C18; 5
micron particle, 120 l~ pore).
Elution rate: 3 mL/min; Detection: UV, ~, = 220 nm.
Eluent A:O.1 % aq. TFA; Eluent B: Acetonitrile (0.1 % TFA).
Example I - Figures 1A-B
Synthesis of L62
[00234] Summary: As shown in Figures 1A-B, L62 was prepared using the
following
steps: Hydrolysis of (313,513)-3-aminocholan-24-oic acid methyl ester A with
NaOH gave the
corresponding acid B, which was then reacted with Fmoc-CI to give intermediate
C. Rink
amide resin functionalised with the octapeptide Gln-Trp-Ala-Val-Gly-His-Leu-
Met-NH2
(BBN[7-14] [SEQ ID NO:1]) was sequentially reacted with C, Fmoc-glycine and
DOTA tri-
t-butyl ester. After cleavage and deprotection with Reagent B the crude was
purified by
preparative HPLC to give L62. Overall yield: 2.5%. More details are provided
below:
A. Rink amide resin functionalised with Bombesin[7-141, (A)
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[00235] In a solid phase peptide synthesis vessel (see enclosure No. 1) Fmoc-
aminoacid (24 mmol), N hydroxybenzotriazole (HOBt) (3.67 g; 24 mmol), and N,N'-
diisopropylcarbodiimide (DIC) (3.75 mL; 24 mmol) were added sequentially to a
suspension
of Rink amide NovaGelTM resin (10 g; 6.0 mmol) A in DMF (45 mL). The mixture
was
shaken for 3 h at room temperature using a bench top shaker, then the solution
was emptied
and the resin was washed with DMF (5 x 45 mL). The resin was shaken with 25%
piperidine
in DMF (45 mL) for 4 min, the solution was emptied and fresh 25% piperidine in
DMF (45
mL) was added. The suspension was shaken for 10 min, then the solution was
emptied and
the resin was washed with DMF (5 x 45 mL).
[00236] This procedure was applied sequentially for the following amino acids:
N a-
Fmoc-L-methionine, N oc-Fmoc-L-leucine, N a,-Fmoc-Nm-trityl-L-histidine, N a.-
Fmoc-
glycine, N a-Fmoc-L-valine, N oc-Fmoc-L-alanine, N a.-Fmoc-N"'-Boc-L-
tryptophan.
[00237] In the last coupling reaction N a-Fmoc-N y-trityl-L-glutamine (14.6 g;
24
mmol), HOBt (3.67 g; 24 mmol), and DIC (3.75 mL; 24 mmol) were added to the
resin in
DMF (45 mL). The mixture was shaken for 3 h at room temperature, the solution
was
emptied and the resin was washed with DMF (5 x 45 mL}, CH2Cl2 (5 x 45 mL) and
vacuum
dried.
B. Preparation of intermediates B and C (FIG 1A):
Synthesis of (313,513)-3-Aminocholan-24-oic acid (B)
[00238] A 1 M solution ofNaOH (16.6 mL; 16.6 mrnol) was added dropwise to
a solution of (313,513)-3-aminocholan-24-oic acid methyl ester (5.0 g;
12.8 mmol) in MeOH (65 mL) at 45 °C. After 3 h stirring at 45
°C, the
mixture was concentrated to 25 mL and H20 (40 mL) and 1 M HCl
(22 mL) were added. The precipitated solid was filtered, washed with
H20 (2 x 50 mL) and vacuum dried to give B as a white solid (5.0 g;
13.3 mmol). Yield 80%.
2. Synthesis of (313,513, -L(9H Fluoren-9-ylmethoxy,)aminocholan-24-oic
acid C
[00239] A solution of 9-fluorenylmethoxycarbonyl chloride (0.76 g; 2.93
mmol) in 1,4-dioxane (9 mL) was added dropwise to a suspension of
(313,513)-3-aminocholan-24-oic acid B (1.0 g; 2.66 mmol) in 10% aq.
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73
Na2CO3 (16 mL) and 1,4-dioxane (9 mL) stirred at 0 °C. After 6 h
stirring at room temperature H20 (90 mL) was added, the aqueous
phase washed with Et20 (2 x 90 mL) and then 2 M HCI (15 mL) was
added (final pH: 1.5). The aqueous phase was extracted with EtOAc (2
x 100 mL), the organic phase dried over Na2S04 and evaporated. The
crude was purified by flash chromatography to give C as a white solid
(1.2 g; 2.0 mmol). Yield 69%.
C. Synthesis ofL62 (N [(313,513)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-
tetraazacyclododec-1-yl]acetyl]amino] acetyl]amino]-cholan-24-yl]-L-
glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-
methioninamide) (FIG 1B):
[00240] Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide
synthesis vessel
with 50% morpholine in DMA (7 mL) for 10 min, the solution was emptied
and fresh 50% morpholine in DMA (7 mL) was added. The suspension was
shaken for 20 min then the solution was emptied and the resin washed with
DMA (5 x 7 mL). (313,SJ3)-3-(9H Fluoren-9-ylmethoxy)aminocholan-24-oic
acid C (0.72 g; 1.2 mmol), N hydroxybenzotriazole (HOBt) (0.18 g; 1.2
mmol), N,N'-diisopropylcarbodiimide (DIC) (0.19 mL; 1.2 mmol) and DMA
(7 mL) were added to the resin, the mixture shaken for 24 h at room
temperature, and the solution was emptied and the resin washed with DMA (5
x 7 mL). The resin was then shaken with 50% morpholine in DMA (7 mL) for
10 min, the solution was emptied, fresh 50% morpholine in DMA (7 mL) was
added and the mixture shaken for another 20 min. The solution was emptied
and the resin washed with DMA (5 x 7 mL). N a-Fmoc-glycine (0.79 g; 1.2
mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol) and DMA (7 mL)
were added to the resin. The mixture was shaken for 3 h at room temperature,
the solution was emptied and the resin washed with DMA (5 x 7 mL). The
resin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the
solution was emptied, fresh 50% morpholine in DMA (7 mL) was added and
the mixture shaken for another 20 min. The solution was emptied and the
resin washed with DMA (5 x 7 mL) followed by addition of 1,4,7,10-
tetraazacyclododecane-1,4,7,10-tetraacetic acid tris(1,1-dimethylethyl) ester
adduct with NaCI (0.79 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19
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mL: 1.2 mmol), DIEA (0.40 mL; 2.4 mmol) and DMA (7 mL) to the resin.
The mixture was shaken for 24 h at room temperature, the solution was
emptied and the resin washed with DMA (5 x 7 mL), CH2C12 (5 x 7 mL) and
vacuum dried. The resin was shaken in a flask with Reagent B (25 mL) for
4.5 h. The resin was filtered and the solution was evaporated under reduced
pressure to afford an oily crude which was triturated with Et2O (20 mL) gave
a precipitate. The precipitate was collected by centrifugation and washed with
Et2O (3 x 20 mL), then analysed by HPLC and purified by preparative HPLC.
The fractions containing the product were lyophilised to give L62 (6.6 mg; 3.8
x 10-3 mmol) as a white solid. Yield 4.5%.
Example II - Figures 2A-F
Synthesis of L70, L73, L74, L115 and L116
[00241] Summary: The products were obtained by coupling of the octapeptide Gln-
Trp-
Ala-Val-Gly-His-Leu-Met-NH2 (BBN[7-14] [SEQ ID NO:l]) (with appropriate side
chain
protection) on the Rink amide resin with different linkers, followed by
functionalization with
DOTA tri-t-butyl ester. After cleavage and deprotection with Reagent B the
final products
were purified by preparative HPLC. Overall yields 3-9%.
A. Synthesis of L70 (FIG. 2A~:
[00242] Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide
synthesis vessel
with 50% morpholine in DMA (7 mL) for 10 min, the solution was emptied
and fresh 50% morpholine in DMA (7 mL) was added. The suspension was
stirred for 20 min then the solution was emptied and the resin washed with
DMA (5 x 7 mL). Fmoc-4-aminobenzoic acid (0.43 g; 1.2 mmol), HOBt (0.18
g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol) and DMA (7 mL) were added to the
resin, the mixture shaken for 3 h at room temperature, the solution was
emptied and the resin washed with DMA (5 x 7 mL). The resin was then
shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution was
emptied, fresh 50% morpholine in DMA (7 mL) was added and the mixture
was shaken fox 20 min. The solution was emptied and the resin washed with
DMA (5 x 7 mL). Fmoc-glycine (0.36 g; 1.2 mmol) HATU (0.46 g; 1.2
mmol) and DIEA (0.40 mL; 2.4 mmol) were stirred for 15 min in DMA (7
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mL) then the solution was added to the resin, the mixture shaken for 2 h at
room temperature, the solution was emptied and the resin washed with DMA
(5 x 7 mL). The resin was then shaken with 50% morpholine in DMA (7 mL)
for 10 min, the solution was emptied, fresh 50% morpholine in DMA (7 mL)
5 was added and the mixture shaken for 20 min. The solution was emptied and
the resin washed with DMA (5 x 7 mL). 1,4,7,10-Tetraazacyclododecane-
1,4,7,10-tetraacetic acid tris(l,l-dimethylethyl) ester adduct with NaCI (0.79
g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), DIEA
(0.40 mL; 2.4 mmol) and DMA (7 mL) were added to the resin. The mixture
10 was shaken for 24 h at room temperature, the solution was emptied and the
resin washed with DMA (5 x 7 mL), CH2C12 (5 x 7 mL) and vacuum dried.
The resin was shaken in a flask with Reagent B (25 mL) for 4 h. The resin
was filtered and the filtrate solution was evaporated under reduced pressure
to
afford an oily crude that was triturated with Et20 (5 mL). The precipitate was
15 collected by centrifugation and washed with Et2O (5 x 5 mL), then analysed
by HPLC and purified by preparative HPLC. The fractions containing the
product were lyophilised to give L70 as a white fluffy solid (6.8 mg; 0.005
mmol). Yield 3%.
B. Synthesis of L73, L115 and L116 (FIGS. 2B - 2E):
20 [00243] Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide
synthesis vessel
with 50% morpholine in DMA (7 mL) for 10 min, the solution was emptied
and fresh 50% morpholine in DMA (7 mL) was added. The suspension was
stirred for 20 min then the solution was emptied and the resin washed with
DMA (5 x 7 mL). Fmoc-linker-OH (1.2 mmol), HOBt (0.18 g; 1.2 mmol),
25 DIC (0.19 mL; 1.2 mmol) and DMA (7 mL) were added to the resin, the
mixture was shaken for 3 h at room temperature, the solution was emptied and
the resin was washed with DMA (5 x 7 mL). The resin was shaken with 50%
morpholine in DMA (7 mL) for 10 min, the solution was emptied, fresh 50%
morphoIine in DMA (7 mL) was added and the mixture was shaken for 20
30 min. The solution was emptied and the resin washed with DMA (5 x 7 mL).
1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid tris(l,l-
dimethylethyl) ester adduct with NaCI (0.79 g; 1.2 mmol), HOBt (0.18 g; 1.2
mmol), DIC (0.19 mL: 1.2 mmol), DIEA (0.40 mL; 2.4 mmol) and DMA (7
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mL) were added to the resin. The mixture was shaken for 24 h at room
temperature, the solution was emptied and the resin washed with DMA (5 x 7
mL), CH2Cl2 (5 x 7 mL) and vacuum dried. The resin was shaken in a flask
with Reagent B (25 mL) for 4 h. The resin was filtered and the solution was
evaporated under reduced pressure to afford an oily crude that was triturated
with Et20 (5 mL). The precipitate was collected by centrifugation and
washed with Et20 (5 x 5 mL), then analysed by HPLC and purified by
preparative HPLC. The fractions containing the product were lyophilised.
C. Synthesis of L74 (FIG. 2F):
[00244 Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide synthesis
vessel
with 50% morpholine in DMA (7 mL) for 10 min, the solution was emptied
and fresh 50% morpholine in DMA (7 mL) was added. The suspension was
stirred for 20 min then the solution was emptied and the resin was washed
with DMA (5 x 7 mL). Fmoc-isonipecotic acid (0.42 g; 1.2 mmol), HOBt
(0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol) and DMA (7 mL) were added to
the resin, the mixture was shaken for 3 h at room temperature, the solution
was
emptied and the resin was washed with DMA (5 x 7 mL). The resin was
shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution was
emptied, fresh 50% morpholine in DMA (7 mL) was added and the mixture
was shaken for 20 min. The solution was emptied and the resin was washed
with DMA (5 x 7 mL). Fmoc-glycine (0.36 g; 1.2 mmol), HOBt (0.18 g; 1.2
mrnol), DIC (0.19 mL; 1.2 mmol) and DMA (7 mL) were added to the resin,
the mixture was shaken for 3 h at room temperature, the solution was emptied
and the resin washed with DMA (5 x 7 mL). The resin was then shaken with
50% morpholine in DMA (7 mL) for 10 min, the solution was emptied, fresh
50% morpholine in DMA (7 mL) was added and the mixture shaken for 20
minutes. The solution was emptied and the resin was washed with DMA (5 x
7 mL). 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid tris(1,1-
dimethylethyl) ester adduct with NaCl (0.79 g; 1.2 mmol), HOBt (0.18 g; 1.2
mmol), DIC (0.19 mL: 1.2 mmol), DIEA (0.40 mL; 2.4 mmol) and DMA (7
mL) were added to the resin. The mixture was shaken for 24 h at room
temperature, the solution was emptied and the resin was washed with DMA (5
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x 7 mL), CH2C12 (5 x 7 mL) and vacuum dried. The resin was shaken in a
flask with Reagent B (25 mL) for 4 h. The resin was filtered and the solution
was evaporated under reduced pressure to afford an oily crude that was
triturated with Et20 (5 mL). The precipitate was collected by centrifugation
and washed with Et20 (5 x 5 mL), then analysed by HPLC and purified by
HPPLC. The fractions containing the product were lyophilised to give L74 as
a white fluffy solid (18.0 mg; 0.012 mmol). Yield 8%.
Example III - Figures 3A-E
Synthesis of L67
[00245] Summary: Hydrolysis of (313,513)-3-amino-12-oxocholan-24-oic acid
methyl ester
A with NaOH gave the corresponding acid B, which was then reacted with Fmoc-
Glycine to
give intermediate C. Rink amide resin functionalised with the octapeptide Gln-
Trp-Ala-Val-
Gly His-Leu-Met-NH2 (BBN[7-14] [SEQ ID NO:1]) was sequentially reacted with C,
and
DOTA tri-t-butyl ester. After cleavage and deprotection with Reagent B the
crude was
purified by preparative HPLC to give L67. Overall yield: 5.2%.
A. Synthesis (313,513)-3-Amino-12-oxocholan-24-oic acid (B) (FIG 3A)
[00246] A 1 M solution of NaOH (6.6 mL; 6.6 mmol) was added dropwise to a
solution of (313,513)-3-amino-12-oxocholan-24-oic acid methyl ester A (2.1 g;
5.1 mmol) in MeOH (15 mL) at 45 °C. After 3 h stirring at 45 °C,
the mixture
was concentrated to 25 mL then H20 (25 mL) and 1 M HCI (8 mL) were
added. The precipitated solid was filtered, washed with H20 (2 x 30 mL) and
vacuum dried to give B as a white solid (1.7 g; 4.4 mmol). Yield 88%.
B. Synthesis of (313,5131-3-[[(9H Fluoren-9-ylmethoxy, amino]'acetyl]'amino-12-
oxocholan-24-oic acid ~C) (FIG 3A)
[00247] Tributylamine (0.7 mL; 3.1 mmol) was added dropwise to a solution ofN
a-
Fmoc-glycine (0.9 g; 3.1 mmol) in THF (25 mL) stirred at 0 °C.
Isobutyl
chloroformate (0.4 mL; 3.1 mmol) was subsequently added and, after 10 min,
a suspension of tributylamine (0.6 mL; 2.6 mmol) and (3J3,513)-3-amino-12-
oxocholan-24-oic acid B (1.0 g; 2.6 mmol) in DMF (30 mL) was added
dropwise, over 1 h, into the cooled solution. The mixture was allowed to
warm up and after 6 h the solution was concentrated to 40 mL, then H20 (50
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mL) and 1 N HC1 (10 mL) were added (final pH: 1.5). The precipitated solid
was filtered, washed with H20 (2 x 50 mL), vacuum dried and purified by
flash chromatography to give C as a white solid (1.l g; 1.7 mmol). Yield
66%.
C. Synthesis of L67 (N ((313 513)-3-[~[f f4 7 10-Tris(carbox methyl)-1 4 7 10-
tetraazacyclododec-1-~)acetyllamino) acet~]'aminol-12 24-dioxocholan-24-
yll-L-~lutaminyl-L-try .~~tophyl-L-alanyl-L-val~l-~lycyl-L-hi stidyl-L-leuc.
methioninamide) (FIG 3B and FIG 3E).
[00248) Resin D (0.5 g; 0.3 mmol) was shaken in a solid phase peptide
synthesis vessel
with 50% morpholine in DMA (7 mL) for 10 min, the solution was emptied
and fresh 50% morpholine in DMA (7 mL) was added. The suspension was
stirred for 20 min then the solution was emptied and the resin was washed
with DMA (5 x 7 mL). (313,513)-3-[[(9H Fluoren-9-
ylmethoxy)amino)acetyl)amino)-12-oxocholan-24-oic acid C (0.80 g; 1.2
mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol) and DMA (7 mL)
were added to the resin, the mixture was shaken for 24 h at room temperature,
the solution was emptied and the resin was washed with DMA (5 x 7 mL).
The resin was shaken with 50% morpholine in DMA (7 mL) for 10 min, the
solution was emptied, fresh 50% morpholine in DMA (7 mL) was added and
the mixture was shaken for 20 min. The solution was emptied and the resin
was washed with DMA (5 x 7 mL). 1,4,7,10-Tetraazacyclododecane-1,4,7,10-
tetraacetic acid tris(1,1-dimethylethyl) ester adduct with NaCI (0.79 g; 1.2
mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), DIEA (0.40 mL;
2.4 mmol) and DMA (7 mL) were added to the resin. The mixture was shaken
for 24 h at room temperature, the solution was emptied and the resin was
washed with DMA (5 x 7 mL), CH2Cl2 (5 x 7 mL) and vacuum dried. The
resin was shaken in a flask with Reagent B (25 mL) for 4.5 h. The resin was
filtered and the solution was evaporated under reduced pressure to afford an
oily crude that was triturated with Et20 (20 mL).
Example IV - Figures 4A-H
Synthesis of L63 and L64
[00249) Summary: Hydrolysis of (313,513,7a,12a)-3-amino-7,12-dihydroxychoIan-
24-oic
acid methyl ester 1b with NaOH gave the intermediate 2b, which was then
reacted with
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Fmoc-glycine to give 3b. Rink amide resin functionalised with the octapeptide
Gln-Trp-Ala-
Val-Gly-His-Leu-Met-NH2 (BBN[7-14] [SEQ ID NO:1 )) was reacted with 3b and
then with
DOTA tri-t-butyl ester. After cleavage and deprotection with Reagent B the
crude was
purified by preparative HPLC to give L64. The same procedure was repeated
starting from
intermediate Za, already available, to give L63. Overall yields: 9 and 4%,
respectively.
A. Synthesis of (313,513,7a,12a)-3-Amino-7,12-dihydroxycholan-24-oic acid,
(2b)
(FIG. 4A1
[00250] A 1 M solution of NaOH (130 mL; 0.13 mol) was added dropwise to a
solution of (3>3,513,7a,12a)-3-amino-7,12-dihydroxycholan-24-oic acid methyl
ester 1b (42.1 g; 0.10 mol) in MeOH (300 mL) heated at 45 °C. After 3 h
stirring at 45°C, the mixture was concentrated to 150 mL and H20 (350
mL)
was added. After extraction with CH2C12 (2 x 100 mL) the aqueous solution
was concentrated to 200 mL and 1 M HCl (150 mL) was added. The
precipitated solid was filtered, washed with H20 (2 x 100 mL) and vacuum
dried to give 2b as a white solid (34.8 g; 0.08 mol). Yield 80%.
B. Synthesis of (313,513,12a)-3-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-
12-h droxycholan-24-oic acid, (3a~(FIG. 4A)
[00251] Tributylamine (4.8 mL; 20.2 mmol) was added dropwise to a solution ofN-
a-
Fmoc-glycine (6.0 g; 20.2 mmol) in THF (120 mL) stirred at 0°C.
Isobutyl
chloroformate (2.6 mL; 20.2 mmol) was subsequently added and, after 10 min,
a suspension of tributylamine (3.9 mL; 16.8 mmol) and (313,513,12a)-3-amino-
12-hydroxycholan-24-oic acid 2a (6.6 g; 16.8 mmol) in DMF (120 mL) was
added dropwise, over 1 h, into the cooled solution. The mixture was allowed
to warm up and after 6 h the solution was concentrated to 150 mL, then H20
(250 mL) and 1 N HCI (40 mL) were added (final pH: 1.5). The precipitated
solid was filtered, washed with H20 (2 x 100 mL), vacuum dried and purified
by flash chromatography to give 3a as a white solid (3.5 g; 5.2 mmol). Yield
31 %.
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C. Synthesis of (313,513,7a,12a)-3-[[(9H-Fluoren-9-
ylmethoxy)amino]acetyl]amino-7 12-dihydroxycholan-24-oic acid (3b) (FIG
[00252) Tributylamine (3.2 mL; 13.5 mmol) was added dropwise to a solution of
N-a-
5 Fmoc-glycine (4.0 g; 13.5 mmol) in THF (80 mL) stirred at 0°C.
Isobutyl
chloroformate (1.7 mL; 13.5 mmol) was subsequently added and, after 10 min,
a suspension of tributylamine (2.6 mL; 11.2 mmol) and (3J3,513,7a,12a)-3-
amino-7,12-dihydroxycholan-24-oic acid 3a (4.5 g; 11.2 mmol) in DMF (80
mL) was added dropwise, over 1 h, into the cooled solution. The mixture was
I 0 allowed to warm up and after 6 h the solution was concentrated to 120 mL,
then H20 (180 mL) and 1 N HCl (30 mL) were added (final pH: 1.5). The
precipitated solid was filtered, washed with H20 (2 x 100 mL), vacuum dried
and purified by flash chromatography to give 3a as a white solid (1.9 g; 2.8
mmol). Yield 25%.
15 [00253] In an alternative method, (313,513,7a,12a)-3-[[(9H-Fluoren-9-
ylmethoxy)amino]acetyl]amino-7,12-dihydroxycholan-24-oic acid, (3b) can
be prepared as follows:
[00254] N-Hydroxysuccinimide (1.70 g, 14.77 mmol) and DIC (1.87 g, 14.77 mmol)
were added sequentially to a stirred solution of Fmoc-Gly-OH (4.0 g, 13.45
20 mmol) in dichloromethane (15 mL); the resulting mixture was stirred at room
temperature for 4 h. The N,N'-diisopropylurea formed was removed by
filtration and the solid was washed with ether (20 mL). The volatiles were
removed and the solid Fmoc-Gly-succinimidyl ester formed was washed with
ether (3 x 20 mL). Fmoc-Gly-succinimidyl ester was then redissolved in dry
25 DMF (15 mL) and 3-aminodeoxycholic acid (5.21 g, 12.78 mmol) was added
to the clear solution. The reaction mixture was stirred at room temperature
for
4 h, water (200 mL) was added and the precipitated solid was filtered, washed
with water, dried and purified by silica gel chromatography (TLC (silica):
(Rf:
0.50, silica gel, CHZCI2/CH30H, 9:1) (eluant: CH2Cla/CH30H (9:1)) to give
30 (313,513,7a,12a)-3-[[(9H-Fluoren-9-ylmethoxy)amino)acetyl]amino-7,12-
dihydroxycholan-24-oic acid as a colorless solid. Yield: 7.46 g (85 %).
D. Synthesis ofL63 (N [(313,513,12a)-3-[[[[[4,7,10-Tris(carboxymethyl)-
1,4,7,10
tetraazacyclododec-1-yl] acetyl]amino]acetyl]amino]-12-hydroxy-24
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oxocholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-
histidyl-L-leucyl-L-methioninamide~FIG. 4B)
[00255) Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide
synthesis vessel
with SO% morpholine in DMA (7 mL) for 10 min, the solution was emptied
and fresh SO% morpholine in DMA (7 mL) was added. The suspension was
stirred for 20 min then the solution was emptied and the resin washed with
DMA (5 x 7 mL). (313,5J3,12a)-3-[[(9H-Fluoren-9-
ylmethoxy)amino]acetyl]amino-12-hydroxycholan-24-oic acid 3a (0.82 g; 1.2
mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol) and DMA (7 mL)
were added to the resin, the mixture was shaken for 24 h at room temperature,
the solution was emptied and the resin was washed with DMA (5 x 7 mL).
The resin was then shaken with SO% morpholine in DMA (7 mL) for 10 min,
the solution was emptied, fresh 50% morpholine in DMA (7 mL) was added
and the mixture was shaken for 20 min. The solution was emptied and the
resin washed with DMA (S x 7 mL). 1,4,7,10-Tetraazacyclododecane-
1,4,7,10-tetraacetic acid tris(l,l-dimethylethyl) ester adduct with NaCI (0.79
g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), DIEA
(0.40 mL; 2.4 mmol) and DMA (7 mL) were added to the resin. The mixture
was shaken for 24 h at room temperature, the solution was emptied and the
resin washed with DMA (5 x 7 mL), CH2C12 (5 x 7 mL) and vacuum dried.
The resin was shaken in a flask with Reagent B (25 mL) for 4 h. The resin
was filtered and the solution was evaporated under reduced pressure to afford
an oily crude that after treatment with Et20 (5 mL) gave a precipitate. The
precipitate was collected by centrifugation and washed with Et20 (5 x S mL),
then analysed and purified by HPLC. The fractions containing the product
were lyophilised to give L63 as a white fluffy solid (12.8 mg; 0.0073 mmol).
Yield 9%.
E. Synthesis ofL64 (N [(313,513,7a,12a)-3-[[[[[4,7,10-Tris(carboxymethyl)-
1,4,7,10-tetraazacyclododec-1-yl] acetyl]amino]acetyl]amino]-7,12-
3 0 dihydroxy-24-oxo chol an-24-yl]-L-glutaminyl-L-tryptophyl-L-al anyl-L-
valyl
glycyl-L-histidyl-L-leucyl-L-methioninamide) (FIG 4C)
[00256] Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide
synthesis vessel
with 50% morpholine in DMA (7 mL) for 10 min, the solution was emptied
and fresh 50% morpholine in DMA (7 mL) was added. The suspension was
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82
stirred for 20 min, the solution was emptied and the resin was washed with
DMA (5 x 7 mL). (313,513,7a,12a)-3-[[(9H-Fluoren-9-
ylmethoxy)amino]acetyl]amino-7,12-dihydroxycholan-24-oic acid 3b (0.81 g;
1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol) and DMA (7
mL) were added to the resin, the mixture was shaken for 24 h at room
temperature, the solution was emptied and the resin was washed with DMA (5
x 7 mL). The resin was shaken with 50% morpholine in DMA (7 mL) for 10
min, the solution was emptied, fresh 50% morpholine in DMA (7 mL) was
added and the mixture was shaken for 20 min. The solution was emptied and
the resin was washed with DMA (5 x 7 mL). 1,4,7,10-
Tetraazacyclododecane-1,4,7,10-tetraacetic acid tris(1,1-dimethylethyl) ester
adduct with NaCI (0.79 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19
mL: 1.2 mmol), DIEA (0.40 mL; 2.4 mmol) and DMA (7 mL) were added to
the resin. The mixture was shaken for 24 h at room temperature, the solution
was emptied and the resin washed with DMA (5 x 7 mL), CH2Cl2 (S x 7 mL)
and vacuum dried. The resin was shaken in a flask with Reagent B (25 mL)
for 4 h. The resin was filtered and the solution was evaporated under reduced
pressure to afford an oily crude that was triturated with Et20 (5 mL). The
precipitate was collected by centrifugation and washed with Et20 (5 x 5 mL).
Then it was dissolved in H20 (20 mL), and Na2C03 (0.10 g; 0.70 mmol) was
added; the resulting mixture was stirred 4 h at room temperature. This
solution was purified by HPLC, the fractions containing the product
lyophilised to give L64 as a white fluffy solid (3.6 mg; 0.0021 mmol). Yield
4%.
Example V - Figures SA-E
Synthesis of L71 and L72
[00257] Summary: The products were obtained in two steps. The first step was
the solid
phase synthesis of the octapeptide GIn-Trp-Ala-Val-Gly-His-Leu-Met-NH2 (BBN[7-
14]
[SEQ ID NO:1]) (with appropriate side chain protecting groups) on the Rink
amide resin
discussed supra. The second step was the coupling with different linkers
followed by
functionalization with DOTA tri-t-butyl ester. After cleavage and deprotection
with Reagent
B the final products were purified by preparative HPLC. Overall yields 3-9%.
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83
A. Bombesin [7-14]' functionalisation and cleavage procedure (FIGS SA and 5D)
[0025] The resin B (0.5 g; 0.3 mmol) was shaken in a solid phase peptide
synthesis
vessel with 50% morpholine in DMA (7 mL) for 10 min, the solution was
emptied and fresh 50% morpholine in DMA (7 mL) was added. The
suspension was stirred for 20 min then the solution was emptied and the resin
was washed with DMA (5 x 7 mL). The Fmoc-linker-OH (1.2 mmol), HOBt
(0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol) and DMA (7 mL) were added to
the resin. The mixture was shaken for 3 h at room temperature, the solution
was emptied and the resin washed with DMA (5 x 7 mL). The resin was then
shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution was
emptied, fresh 50% morpholine in DMA (7 mL) was added and the mixture
was shaken for 20 min. The solution was emptied and the resin was washed
with DMA (5 x 7 mL). 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic
acid tris(l,l-dimethylethyl) ester adduct with NaCI C (0.79 g; 1.2 mmol),
HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), DIEA (0.40 mL; 2.4
mmol) and DMA (7 mL) were added to the resin. The mixture was shaken for
24 h at room temperature. The solution was emptied and the resin washed
with DMA (5 x 7 mL), CH2Cl2 (5 x 7 mL) and vacuum dried. The resin was
shaken in a flask with Reagent B (25 mL) for 4 h. The resin was filtered and
the filtrate was evaporated under reduced pressure to afford an oily crude
that
was triturated with ether (5 mL). 'The precipitate was collected by
centrifugation and washed with ether (5 x 5 mL), then analyzed by analytical
HPLC and purified by preparative HPLC. The fractions containing the
product were lyophilized.
B. Products
[00259] 1. L71 (4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-
y1] acetyl] amino]methyl]benzoyl-L-glutaminyl-L-tryptophyl-L-al anyl-
L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide)
The product was obtained as a white fluffy solid (7.3 mg; 0.005
mmol). Yield 7.5%.
[00260] 2. L72 (Trans-4-[[[[4,7,10-tris(carboxymethyl)-1,4,7,10-
tetraazacyclododec-1-yl]acetyl]amino]methyl]cyclohexylcarbonyl-L-
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glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycil-L-histidyl-L-leucyl-
L-methioninamide)
The product was obtained as a white fluffy solid (7.0 mg; 0.005
mmol). Yield 4.8%.
S C. Ttrans-4-[[[(9H-fluoren-9-
ylmethoxy)carbonyl-aminoJmethyl]''cyclohexanecarboxylic acid (D) (FIG.
SE
[00261] A solution ofN (9-fluorenylmethoxycarbonyloxy)succinimide (4.4 g; 14.0
mmol) in 1,4-dioxane (40 mL) was added dropwise to a solution of tr-ans-4-
(aminomethyl)cyclohexanecarboxylic acid (2.0 g; 12.7 mmol) in 10%
Na2C03 (30 mL) cooled to 0 °C. The mixture was then allowed to
warm to
ambient temperature and after 1 h stirring at room temperature was treated
with 1 N HCl (32 mL) until the final pH was 2. The resulting solution was
extracted with n-BuOH (100 mL); the volatiles were removed and the crude
residue was purified by flash chromatography to give D as a white solid (1.6
g; 4.2 mmol). Yield 33%.
Example VI - Figures 6A-F
Synthesis of L75 and L76
[00262] Summary: The two products were obtained by coupling of the octapeptide
Gln-
Trp-Ala-Val-Gly-His-Leu-Met-NH2 (BBN[7-14] [SEQ ID N0:1]) (A) on the Rink
amide
resin with the two linkers E and H, followed by functionalization with DOTA
tri-t-butyl ester.
After cleavage and deprotection with Reagent B the final products were
purified by
preparative HPLC. Overall yields: 8.5% (L75) and 5.6% (L76).
A. 2-[(1,3-Dihydro-1,3-dioxo-2H isoindol-2-~lmethy~'benzoic acid's (FIG.
6A
[00263] The product was synthesized following the procedure reported in the
literature
(Bornstein, J; Drummon, P. E.; Bedell, S. F. Org Synth. Coll. Vol. IV 1963,
810-812).
B. 2-(Aminomethyl)benzoic acid, (D~(FIG. 6A~
[00264] A 40% solution ofmethylamine (6.14 mL; 7.lmmol) was added to 2-[(1,3-
dihydro-1,3-dioxo-2H isoindol-2-yl)methyl]benzoic acid C (2 g; 7.1 mmol)
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and then EtOH (30 mL) was added. After 5 minutes stirring at room
temperature the reaction mixture was heated at 50 °C. After 2.5 h, the
mixture
was cooled and the solvent was evaporated under reduced pressure. The crude
product was suspended in 50 mL of absolute ethanol and the suspension was
S stirred at room temperature for 1 h. The solid was filtered and washed with
EtOH to afford 2-(aminomethyl)benzoic acid D (0.87 g; 5.8 mmol). Yield
81 %.
C. 2-[[[9H Fluoren-9-ylmethoxy carbons amino]meth~lbenzoic acid (E) (FIG.
10 [00265] The product was synthesized following the procedure reported in the
literature
(Sun, J-H.; Deneker, W. F. Synth. Commun. 1998, 28, 4525-4530).
D. 4-(Aminomethyl)-3-nitrobenzoic acid, (G) (FIG. 6B)
[00266] 4-(Bromomethyl)-3-nitrobenzoic acid (3.2 g; 12.3 mmol) was dissolved
in 8%
NH3 in EtOH (300 mL) and the resulting solution was stirred at room
15 temperature. After 22 h the solution was evaporated and the residue
suspended in H20 (70 mL). The suspension was stirred for 15 min and
filtered. The collected solid was suspended in H20 (40 mL) and dissolved by
the addition of few drops of 25% aq. NH40H (pH 12), then the pH of the
solution was adjusted to 6 by addition of 6 N HCl. The precipitated solid was
20 filtered, and washed sequentially with MeOH (3 x 5 mL), and Et20 (10 mL)
and was vacuum dried (1.3 kPa; P205) to give 4-(aminomethyl)-3-
nitrobenzoic acid as a pale brown solid (1.65 g; 8.4 mmol). Yield 68%.
E. 4-[[[9H Fluoren-9-ylmethoxy)carbonyl]amino]methyl]-3-nitrobenzoic acid,
(H) (FIG. 6B)
25 [00267] 4-(Aminomethyl)-3-nitrobenzoic acid G (0.8 g; 4 mmol) was dissolved
in
10% aq. Na2C03 (25 mL) and 1,4-dioxane (10 mL) and the solution was
cooled to 0 °C. A solution of 9-fluorenylmethyl chloroformate (Fmoc-Cl)
(1.06 g; 4 mmol) in 1,4-dioxane (10 mL) was added dropwise for 20 min.
After 2 h at 0-5 °C and 1 h at 10 °C the reaction mixture was
filtered and the
30 solution was acidified to pH 5 by addition of 1 N HCI. The precipitate was
filtered, washed with H20 (2 x 2 mL) dried under vacuum (1.3 kPa; P205) to
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86
give 4-[[[9H fluoren-9-ylmethoxy)carbonyl]amino]methyl]-3-nitrobenzoic
acid as a white solid (1.6 g; 3.7 mmol). Yield 92%.
F. L75 (N [2-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-
tetraazacyclododec-1-yl]acetyl]amino] methyl]benzoyl]-L-
glutaminyl-L-tryptophyl-L-alanyl-L-valyl-g-l~yl-L-histid.1-~ucyl-
L-methioninamide) (FIG. 6C1
[00268] Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide
synthesis vessel
with 50% morpholine in DMA (7 mL) for 10 min, the solution was emptied
and fresh 50% morpholine in DMA (7 mL) was added. The suspension was
stirred for 20 min then the solution was emptied and the resin washed with
DMA (5 x 7 mL). 2-[[[9H Fluoren-9-
ylmethoxy)carbonyl]amino]methyl]benzoic acid, E (0.45 g; 1.2 mmol), N
hydroxybenzotriazole (HOBt) (0.18 g; 1.2 mmol), N,N'-
diisopropylcarbodiimide (DIC) (0.19 mL; 1.2 mmol) and DMA (7 mL) were
added to the resin, the mixture shaken for 24 h at room temperature, the
solution was emptied and the resin was washed with DMA (5 x 7 mL). The
resin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the
solution was emptied, fresh 50% morpholine in DMA (7 mL) was added and
the mixture shaken for 20 min. The solution was emptied and the resin
washed with DMA (5 x 7 mL). 1,4,7,10-Tetraazacyclododecane-1,4,7,10-
tetraacetic acid tris(1,1-dimethylethyl) ester adduct with NaCI (DOTA tri-t-
butyl ester) (0.79 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2
mmol), DIEA (0.40 mL; 2.4 mmol) and DMA (7 mL) were added to the resin.
The mixture was shaken for 24 h at room temperature, the solution was
emptied and the resin was washed with DMA (5 x 7 mL), CH2C12 (5 x 7 mL)
and vacuum dried. The resin was shaken in a flask with Reagent B (25 mL)
for 4.5 h. The resin was filtered and the filtrate was evaporated under
reduced
pressure to afford an oily crude that after treatment with Et20 (20 mL) gave a
precipitate. The resulting precipitate was collected by centrifugation and was
washed with Et20 (3 x 20 mL) to give L75 (190 mg; 0.13 mmol) as a white
solid. Yield 44%.
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G. L76 (N [4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-
yl]acetyl]amino] methyl]-3-nitrobenzoyl]-L-glutaminyl-L-tryptophyl-L-
alanyl-L-valyl-~l~yl-L-histidyl-L-leucyl-L-methioninamide) (FIG 6D~
[00269] Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide
synthesis vessel
with 50% morpholine in DMA (7 mL) for 10 min, the solution was emptied
and fresh 50% morpholine in DMA (7 mL) was added. The suspension was
stirred for 20 min then the solution was emptied and the resin was washed
with DMA (5 x 7 mL). 4-[[[9H Fluoren-9-
ylmethoxy)carbonyl]amino]methyl]-3-nitrobenzoic acid, H (0.50 g;1.2 mmol),
HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol) and DMA (7 mL) were
added to the resin, the mixture was shaken for 24 h at room temperature, the
solution was emptied and the resin was washed with DMA (5 x 7 mL). The
resin was then shaken with 50% morpholine in DMA (7 mL) for I 0 min, the
solution was emptied, fresh 50% morpholine in DMA (7 mL) was added and
the mixture was shaken for 20 min. The solution was emptied and the resin
was washed with DMA (5 x 7 mL). DOTA tri-t-butyl ester (0.79 g; 1.2
mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), DIEA (0.40 mL;
2.4 mmol) and DMA (7 mL) were added to the resin. The mixture was shaken
for 24 h at room temperature, the solution was emptied and the resin was
washed with DMA (5 x 7 mL), CH2Cl2 (5 x 7 mL) and vacuum dried. The
resin was shaken in a flask with Reagent B (25 mL) for 4.5 h. The resin was
filtered and the solution was evaporated under reduced pressure to afford an
oily crude that was triturated with Et20 (20 mL). The precipitate was
collected by centrifugation and was washed with Et20 (3 x 20 mL) to give a
solid (141 mg) which was analysed by HPLC. A 37 mg portion of the crude
was purified by preparative HPLC. The fractions containing the product were
lyophilised to give L76 (10.8 mg; 7.2 x 10-3 mmol) as a white solid. Yield
9%.
Example VII - Figures 7A-C
Synthesis of L124
[00270] Summary: 4-Cyanophenol A was reacted with ethyl bromoacetate and K2C03
in
acetone to give the intermediate B, which was hydrolysed with NaOH to the
corresponding
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88
acid C. Successive hydrogenation of C with H2 and Pt02 at 355 kPa in
EtOH/CHC13 gave
the corresponding aminoacid D, which was directly protected with FmocOSu to
give E. Rink
amide resin functionalised with the octapeptide Gln-Trp-Ala-Val-Gly-His-Leu-
Met-NH2
(BBN[7-14] [SEQ ID NO:l]) was reacted with E and then with DOTA tri-t-butyl
ester. After
cleavage and deprotection with Reagent B the crude was purified by preparative
HPLC to
give L124. Overall yield: 1.3%
A. Synthesis of (4-C~anophenoxy)acetic acid ethyl ester, (B) (FIG. 7A)
[00271 ] The product was synthesized following the procedure reported in the
literature
(Archimbault, P.; LeClerc, G.; Strosberg, A. D.; Pietri-Rouxel, F. PCT Int.
Appl. WO 980005, 1998).
B. Synthesis of (4-CyanophenoxX)acetic acid, (C) (FIG. 7A)
[00272] A 1 N solution of NaOH (7.6 mL; 7.6 mmol) was added dropwise to a
solution
of (4-cyanophenoxy)acetic acid ethyl ester B (1.55 g; 7.6 mmol) in MeOH (15
mL). After 1 h the solution was acidified with 1 N HCI (7.6 mL; 7.6 mmol)
and evaporated. The residue was taken up with water (20 mL) and extracted
with CHC13 (2 x 30 mL). The organic phases were evaporated and the crude
was purified by flash chromatography to give (4-cyanophenoxy)acetic acid C
(0.97 g; 5.5 mmol) as a white solid. Yield 72%.
C. Synthesis of [4-[[[9H Fluoren-9-
ylmethoxX carbon~lamino]meth~lphenoxy]'acetic acid, (E (FIG. 7A)
[00273] Pt02 (150 mg) was added to a solution of (4-cyanophenoxy)acetic acid C
(1.05 g; 5.9 mmol) in EtOH (147 mL) and CHCl3 (3 mL). The suspension
was stirred 30 h under a hydrogen atmosphere (355 kPa; 20 °C). The
mixture
was filtered through a Celite~ pad and the solution evaporated under vacuum.
The residue was purified by flash chromatography to give acid D (0.7 g)
which was dissolved in H20 (10 mL), MeCN (2 mL) and Et3N (0.6 mL) at 0
°C, then a solution ofN (9-fluorenylmethoxycarbonyloxy)succinimide (1.3
g;
3.9 mmol) in MeCN (22 mL) was added dropwise. After stirring 16 h at room
temperature the reaction mixture was filtered and the volatiles were removed
under vacuum. The residue was treated with 1 N HCl (10 mL) and the
precipitated solid was filtered and purified by flash chromatography to give
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89
[4-[[[9H fluoren-9-ylmethoxy)carbonyl]amino]methyl]phenoxy]acetic acid E
(0.56 g; 1.4 mmol) as a white solid. Overall yield 24%.
D. Synthesis ofL124 (N [[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-
tetraazacyclododec-1-yl]acetyl] amino]methyl]phenoxy]acetyl]-L-
glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-
methioninamide)
(FIG. 7B)
[00274] Resin A (480 mg; 0.29 mmol) was shaken in a solid phase peptide
synthesis
vessel with 50% morpholine in DMA (7 mL) for 10 min, the solution was
emptied and fresh 50 % morpholine in DMA (7 mL) was added. The
suspension was stirred for 20 min, the solution was emptied and the resin was
washed with DMA (5 x 7 mL). [4-[[[9H Fluoren-9-
ylmethoxy)carbonyl]amino]methyl]phenoxy]acetic acid E (480 mg; 1.19
mmol), N hydroxybenzotriazole (HOBt) (182 mg; 1.19 mmol), N,N'-
diisopropylcarbodiimide (DIC) (185 ~L; 1.19 mmol) and DMA (7 mL) were
added to the resin, the mixture was shaken for 24 h at room temperature, the
solution was emptied and the resin was washed with DMA (5 x 7 mL). The
resin was then shaken with 50% morpholine in DMA (6 mL) for 10 min, the
solution was emptied, fresh 50% morpholine in DMA (6 mL) was added and
the mixture was shaken for 20 min. The solution was emptied and the resin
was washed with DMA (5 x 7 mL). 1,4,7,10-Tetraazacyclododecane-1,4,7,10-
tetraacetic acid tris(1,1-dimethylethyl) ester adduct with NaCI (750 mg; 1.19
mmol), HOBt (182 mg; 1.19 mmol), DIEA (404 ~.L; 2.36 mmol), DIC (185
~L; 1.19 mmol) and DMA (6 mL) were added to the resin. The mixture was
shaken for 24 h at room temperature, the solution was emptied, the resin was
washed with DMA (2 x 7 mL), CH2Cl2 (5 x 7 mL) and vacuum dried. The
resin was shaken in a flask with Reagent B (25 mL) for 4 h. The resin was
filtered and the filtrate was evaporated under reduced pressure to afford an
oily crude that was triturated with Et20 (5 mL). The precipitate was collected
by centrifugation and washed with Et20 (5 x 5 mL) to give a solid (148 mg)
which was analysed by HPLC. A 65 mg portion of the crude was purified by
preparative HPLC. The fractions containing the product were lyophilised to
give L124 (FIG. 7C) as a white solid (15 mg; 0.01 mmol). Yield 7.9%.
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Example VIII - Figures 8A-C
Synthesis of L125
[00275] Summary: 4-(Bromomethyl)-3-methoxybenzoic acid methyl ester A was
reacted
with NaN3 in DMF to give the intermediate azide B, which was then reduced with
Ph3P and
5 H20 to amine C. Hydrolysis of C with NaOH gave acid D, which was directly
protected
with FmocOSu to give E. Rink amide resin functionalised with the octapeptide
Gln-Trp-Ala-
Val-Gly-His-Leu-Met-NH2 (BBN[7-14] [SEQ ID NO:l]) (A) was reacted with E and
then
with DOTA tri-t-butyl ester. After cleavage and deprotection with Reagent B
the crude was
purified by preparative HPLC to give L125. Overall yield: 0.2%.
10 A. Synthesis of 4-(Azidometh~)-3-methoxybenzoic acid methyl ester., (B~FIG.
8A
[00276] A solution of 4-(bromomethyl)-3-methoxybenzoic acid methyl ester (8 g;
31
mmol) and NaN3(2 g; 31 mmol) in DMF (90 mL) was stirred overnight at
room temperature. The volatiles were removed under vacuum and the crude
15 product was dissolved in EtOAc (50 mL). The solution was washed with
water (2 x 50 mL) and dried. The volatiles were evaporated to provide 4-
(azidomethyl)-3-methoxybenzoic acid methyl ester (6.68 g; 30 mmol). Yield
97%.
B. 4-(Aminometh~)-3-methoxybenzoic acid methyl ester, (C) (FIG. 8A)
20 [00277] Triphenylphosphine (6.06 g; 23 mmol) was added to a solution of (4-
azidomethyl)-3-methoxybenzoic acid methyl ester B (5 g; 22 mmol) in THF
(50 mL): hydrogen evolution and formation of a white solid was observed.
The mixture was stirred under nitrogen at room temperature. After 24 h more
triphenylphosphine (0.6 g; 2.3 mmol) was added. After 24 h the azide was
25 consumed and H20 (10 mL) was added. After 4 h the white solid disappeared.
The mixture was heated at 45 °C for 3 h and was stirred overnight
at room
temperature. The solution was evaporated to dryness and the crude was
purified by flash chromatography to give 4-(aminomethyl)-3-methoxybenzoic
acid methyl ester C (1.2 g; 6.1 mmol). Yield 28%.
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91
C. 4-[[[9H Fluoren-9-ylmethoxy)carbonyl]amino]methyl]-3-
methoxybenzoic acid.~E) (FIG. 8A1
A 1 N solution of NaOH (6.15 mL; 6.14 mmol) was added dropwise to a
solution of 4-(aminomethyl)-3-methoxybenzoic acid methyl ester C (1.2 g;
6.14 mmol) in MeOH (25 mL) heated at 40 °C. After stirring 8 h at 45
°C the
solution was stirred over night at room temperature. A 1 N solution of NaOH
(0.6 mL; 0.6 mmol) was added and the mixture heated at 40°C for 4 h.
The
solution was concentrated, acidified with 1 N HCl (8 mL; 8 mmol), extracted
with EtOAc (2 x 10 mL) then the aqueous layer was concentrated to 15 mL.
This solution (pH 4.5) was cooled at 0°C and Et3N (936 ~.L; 6.75
mmol) was
added (pH 11 ). A solution of N (9-fluorenylmethoxycarbonyloxy)succinimide
(3.04 g; 9 mmol) in MeCN (30 mL) was added dropwise (final pH 9) and a
white solid precipitated. After stirring 1 h at room temperature the solid was
filtered, suspended in 1N HCl (15 mL) and the suspension was stirred for 30
min. The solid was filtered to provide 4-[[[9H fluoren-9-
ylmethoxy)carbonyl]amino]methyl]-3-methoxybenzoic acid E as a white solid
(275 mg; 0.7 mmol).
[00278] The filtrate was evaporated under vacuum and the resulting white
residue was
suspended in 1N HCl (20 mL) and stirred for 30 minutes. The solid was
filtered and purified by flash chromatography to give more acid E (198 mg;
0.5 mmol). Overall yield 20%.
D. L125 (N [4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-
yl]acetyl]amino] methyl]-3-methoxybenzoyl]-L-glutaminyl-L-tryptophyl-L-
alanyl-L-valyl-~lycyl-L-histidyl-L-leucyl-L-methioninamide) (FIG. 8B)
[00279] Resin A (410 mg; 0.24 mmol) was shaken in a solid phase peptide
synthesis
vessel with 50% morpholine in DMA (7 mL) for 10 min, the solution was
emptied and fresh 50 % morpholine in DMA (7 mL) was added. The
suspension was stirred for 20 min then the solution was emptied and the resin
was washed with DMA (5 x 7 mL). 4-[[[9H Fluoren-9-
ylmethoxy)carbonyl]amino]methyl]-3-methoxybenzoic acid E (398 mg; 0.98
mmol), HOBt (151 mg; 0.98 mmol), DIC (154 pL; 0.98 mmol) and DMA (6
mL) were added to the resin; the mixture was shaken for 24 h at room
temperature, the solution was emptied and the resin was washed with DMA (5
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x 7 mL). The resin was then shaken with 50% morpholine in DMA (6 mL) for
min, the solution was emptied, fresh 50% morpholine in DMA (6 mL) was
added and the mixture was shaken for 20 min. The solution was emptied and
the resin washed with DMA (5 x 7 mL). 1,4,7,10-Tetraazacyclododecane-
1,4,7,10-tetraacetic acid tris(l,l-dimethylethyl) ester adduct with NaCI (618
mg; 0.98 mmol), HOBt (151 mg; 0.98 mmol), DIC (154 ~L; 0.98 mmol),
DIEA (333 ~L; 1.96 mmol) and DMA (6 mL) were added to the resin. The
mixture was shaken for 24 h at room temperature, the solution was emptied
and the resin was washed with DMA (5 x 7 mL), CH2Cl2 (5 x 7 mL) and
10 vacuum dried. The resin was shaken in a flask with Reagent B (25 mL) for 4
h. The resin was filtered and the solution was evaporated under reduced
pressure to afford an oily crude that was triturated with Et20 (5 mL). The
resulting precipitate was collected by centrifugation, was washed with Et20 (5
x 5 mL), was analysed by HPLC and purified by preparative HPLC. The
fractions containing the product were lyophilised to give L125 (FIG. 8C) as a
white solid (15.8 mg; 0.011 mmol). Yield 4.4%.
Example IX - Figures 9A - 9D
Synthesis of L146, L233, L234, and L235
[00280] Summary: The products were obtained in several steps starting from the
octapeptide Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2(BBN[7-14]) (A) on the Rink
amide
resin. After final cleavage and deprotection with Reagent B the crudes were
purified by
preparative HPLC to give L146, L233, L234 and L235. Overall yields: 10%, 11 %,
4.5%,
5.7% respectively.
A. 3-[[[(9H Fluoren-9-ylmethoxy)carbonyl]amino]acetyl]aminobenzoic
acid, B.(FIG. 9A)
[00281] A solution of 3-aminobenzoic acid (0.5 g; 3.8 mmol) and N
ethyldiisopropylamine (DIEA) (0.64 mL; 3.8 mmol) in THF (20 mL) was
added dropwise to a solution of Fmoc-glycine chloride (1.2 g; 4.0 mmol) (3)
in THF (10 mL) and CHZCI2 (10 mL). After 24 h stirring at room temperature
1 M HCl (50 mL) was added (final pH: 1.5). The precipitate was filtered,
washed with HZO (2 x 100 mL), vacuum dried and crystallised from
CHC13/CH30H (I:I) to give B as a white solid (0.7 g; 1.6 mmol). Yield 43%.
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B. N [3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-
yl]acetyl]amino]acetyl]amino]benzoyl]-L-glutaminyl-L-tryptophyl-L-
alanyl-L-valyl-~lycyl-L-histi~l-L-leucyl-L-methioninamide L233 (FIG.
9D1
[00282] Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide
synthesis vessel
with 50% morpholine in DMA (7 mL) for 10 min, the solution emptied and
fresh 50% morphoIine in DMA (7 mL) was added.
[00283] The suspension was stirred for another 20 min then the solution was
emptied
and the resin washed with DMA (5 x 7 mL). 3-[[[(9H Fluoren-9-
ylmethoxy)carbonyl]amino]acetyl]aminobenzoic acid, B (0.50 g;1.2 mmol),
HOBt (0.I 8 g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol) and DMA (7 mL) were
added to the resin, the mixture shaken for 6 h at room temperature, emptied
and the resin washed with DMA (5 x 7 mL). The resin was then shaken with
50% morpholine in DMA (7 mL) for 10 min, the solution emptied, fresh 50%
morpholine in DMA (7 mL) was added and the mixture shaken for another 20
min. The solution was emptied and the resin washed with DMA (5 x 7 mL).
DOTA tri-t-butyl ester adduct with NaCl2 (0.79 g; 1.2 mmol) (5), HOBt (0.18
g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), DIEA (0.40 mL; 2.4 mmol) and
DMA (7 mL) were added to the resin. The mixture was shaken for 24 h at
room temperature, emptied and the resin washed with DMA (5 x 7 mL),
CH2Cl2 (5 x 7 mL) and vacuum dried. The resin was shaken in a flask with
Reagent B (25 mL) for 4.5 h. The resin was filtered and the solution was
evaporated under reduced pressure to afford an oily crude that after treatment
with Et20 (20 mL) gave a precipitate. The precipitate was collected by
centrifugation and washed with EtaO (3 x 20 mL) to give a solid (152 mg)
which was analysed by HPLC. An amount of crude (50 mg) was purified by
preparative HPLC. The fractions containing the product were lyophilised to
give L233 (17.0 mg; 11.3 x 10-3 mmol) as a white solid. Yield 11%.
C. N [4-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-
3 0 1-yl] acetyl] amino] acetyl] amino]phenyl acetyl]-L-glutaminyl-L-
tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-
methioninamide. L146 (FIG. 9D)
[00284] Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide
synthesis vessel
with 50% morpholine in DMA (7 mL) for 10 min, the solution filtered and
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fresh 50% morpholine in DMA (7 mL) was added. The suspension was stirred
for another 20 min then the solution was filtered and the resin washed with
DMA (5 x 7 mL). Fmoc-4-aminophenylacetic acid (0.45 g; 1.2 mmol), HOBt
(0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol) and DMA (7 mL) were added to
the resin, the mixture shaken for 6 h at room temperature, filtered and the
resin
washed with DMA (5 x 7 mL). The resin was then shaken with 50%
morpholine in DMA (7 mL) for 10 min, the solution filtered, fresh 50%
morpholine in DMA (7 mL) was added and the mixture shaken for another 20
min. The solution was filtered and the resin washed with DMA (5 x 7 mL).
Fmoc-glycine (0.36 g; 1.2 mmol), HATU (0.46 g; 1.2 mmol) and DIEA (0.40
mL; 2.4 mmol) were stirred for 15 min in DMA (7 mL) then the solution was
added to the resin, the mixture shaken for 2 h at room temperature, filtered
and
the resin washed with DMA (5 x 7 mL). The resin was then shaken with 50%
morpholine in DMA (7 mL) for 10 min, the solution filtered, fresh 50%
morpholine in DMA (7 mL) was added and the mixture shaken for another 20
min. The solution was filtered and the resin washed with DMA (5 x 7 mL).
DOTA tri-t-butyl ester adduct with NaCl (0.79 g; 1.2 mmol), HOBt (0.18 g;
I .2 mmol), DIC (0.19 mL; 1.2 mmol}, DIEA (0.40 mL; 2.4 mmol) and DMA
(7 mL) were added to the resin. The mixture was shaken for 24 h at room
temperature, filtered and the resin washed with DMA (5 x 7 mL), CH2Cl2 (5 x
7 mL) and vacuum dried. The resin was shaken in a flask with Reagent B (25
mL) for 4.5 h. The resin was filtered and the solution was evaporated under
reduced pressure to afford an oily crude that after treatment with Et2O (20
mL)
gave a precipitate. The precipitate was collected by centrifugation and washed
with Et20 (3 x 20 mL) to give a solid (203 mg) which was analysed by HPLC.
An amount of crude (50 mg) was purified by preparative HPLC. The fractions
containing the product were lyophilised to give L146 (11.2 mg; 7.4 x 10-3
mmol) as a white solid. Yield 10%.
D. 6-[[[(9FI Fluoren-9-
ylmethoxy)carbonyl]amino]acetyl]aminonaphthoic
acid, C (FIG. 9B)
[00285] A solution of 6-aminonaphthoic acid (500 mg; 2.41 mmol); and DIEA (410
~L 2.41 mmol) in THF (20 mL) was added dropwise to a solution of Fmoc
glycine chloride (760 mg; 2.41 mmol) in CH2Clz/THF 1:1 (10 mL) and stirred
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at room temperature. After 24 h the solvent was evaporated under vacuum.
The residue was taken up with 0.5 N HCI (50 mL) and stirred for 1 h. The
white solid precipitated was filtered and dried. The white solid was suspended
in methanol (30 mL) and boiled for 5 min, then was filtered to give product C
5 (690 mg; 1.48 mmol). Yield 62%.
E. N [6-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-
1-yl]acetyl]amino]acetyl] amino]naphthoyl]-L-glutaminyl-L-
tryptophyl- L-alanyl-L-val I-y~l~yl-L-histidyl-L-leucyl-L-
methioninamide, L234
10 [00286] Resin A (500 mg; 0.3 mmol) was shaken in a solid phase peptide
synthesis
vessel with 50% morpholine in DMA (7 mL) for 10 min, the solution emptied
and fresh 50 % morpholine in DMA (7 mL) was added. The suspension was
stirred for another 20 min then the solution was emptied and the resin washed
with DMA (5 x 7 mL). 6-[[[(9H Fluoren-9-
15 ylmethoxy)carbonyl]amino]acetyl]aminonaphthoic acid C (560 mg; 1.2
mmol), HOBt (184 mg; 1.2 mmol), DIC (187 ~.L; 1.2 mmol) and DMA (7 mL)
were added to the resin, the mixture shaken for 6 h at room temperature,
emptied and the resin washed with DMA (5 x 7 mL). The resin was then
shaken with 50% morpholine in DMA (6 L) for 10 min, the solution emptied,
20 fresh 50% morpholine in DMA (7 mL) was added and the mixture shaken for
another 20 min. The solution was emptied and the resin washed with DMA (5
x 7 mL). DOTA tri-t-butyl ester adduct with NaCI (757 mg; 1.2 mmol), HOBt
(184 mg; 1.2 mmol), DIC (187 p.L; 1.2 mmol), and DIEA (537 ~.L; 2.4 mmol)
and DMA (7 mL) were added to the resin. The mixture was shaken in a flask,
25 emptied and the resin washed with DMA (2 x 7 mL), CHZCI2 (5 x 7 mL) and
vacuum dried. The resin was shaken in a flask with Reagent B (25 mL) for
4.5 h. The resin was filtrated and the solution was evaporated under reduced
pressure to afford an oil crude that after treatment with Et20 (20 mL) gave a
precipitate. The precipitate was collected by centrifugation and washed with
30 Et20 (3 x 20 mL) to give a solid (144 mg) which was analysed by HPLC. An
amount of crude (54 mg) was purified by preparative HPLC. The fractions
containing the product were lyophilised to give L234 (8 mg; 5.1 x 10-3 mmol)
as a white solid. Yield 4.5%.
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F. 4-[[[[(9H Fluoren-9-
ylmethoxy)carbonyl]amino]acetyl]methylamino]benzoic acid, D
(FIG. 9C)
[00287] A solution of 4-N methylaminonaphthoic acid (500 mg; 3.3 mmol) and
DIEA
(562 ~L 3.3 mmol) in THF (20 mL) was added to a solution of Fmoc-glycine
chloride (1.04 g; 3.3 mmol) in CH2C12/THF 1:1 (10 mL) and stirred at room
temperature. After 24 h the solvent was evaporated under vacuum. The
residue was taken up with 0.5 N HCl (30 mL) and was stirred for 3 h at 0
°C.
The white solid precipitated was filtered and dried. The crude was purified by
flash chromatography to give Compound D (350 mg; 0.81 mmol). Yield 25%.
G. N [4-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-
tetraazacyclododec-1-yl] acetyl] amino] acetyl]
methylamino]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-
valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide, L235 (FIG.
9D)
[00288] Resin A (500 mg; 0.3 mmol) was shaken in a solid phase peptide
synthesis
vessel with 50% morpholine in DMA (7 mL) for 10 min, the solution emptied
and fresh 50 % morpholine in DMA (7 mL) was added. The suspension was
stirred for another 20 min then the solution was emptied and the resin washed
with DMA (5 x 7 mL). 4-[[[[9H-Fluoren-9-
ylmethoxy)carbonyl]amino]acetyl]-N methyl]amino-benzoic acid D (510 mg;
1.2 mmol), HOBt (184 mg; 1.2 mmol), DIC (187 ~L; 1.2 mmol) and DMA (7
mL) were added to the resin, the mixture shaken for 6 h at room temperature,
emptied and the resin washed with DMA (5 x 7 mL). The resin was then
shaken with 50% morpholine in DMA (7mL) for 10 min, the solution emptied,
fresh 50% morpholine in DMA (7 mL) was added and the mixture shaken for
another 20 min. The solution was emptied and the resin washed with DMA (5
x 7 mL). DOTA tri-t-butyl ester adduct with NaCI (757 mg; 1.2 mmol), HOBt
(184 mg; 1.2 mmol), DIC (187 ~.L; 1.2 mmol), and DIEA (537 ~L; 2.4 mmol)
and DMA (7 mL) were added to the resin. The mixture was shaken in a flask,
emptied and the resin washed with DMA (2 x 7 mL), CH2CI2 (5 x 7 mL) and
vacuum dried. The resin was shaken in a flask with Reagent B (25 mL) for
4.5 h. The resin was filtrated and the solution was evaporated under reduced
pressure to afford an oil crude that after treatment with Et20 (20 mL) gave a
precipitate.
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[00289] The precipitate was collected by centrifugation and washed with Et20
(3 x 20
mL) to give a solid (126 mg) which was analysed by HPLC. An amount of
crude (53 mg) was purified by preparative HPLC. The fractions containing
the product were lyophilised to give L235 (11 mg; 7.2 x 10-3 mmol) as a white
solid. Yield 5.7%.
EXAMPLE X - Figures l0A-B
Synthesis of L237
[00290] Summary: 1-Formyl-1,4,7,10-tetraazacyclododecane (A) was selectively
protected with benzyl chloroformate at pH 3 to give B, which was alkylated
with t-butyl
bromoacetate and deformylated with hydroxylamine hydrochloride to give D.
Reaction with
P(OtBu)3 and paraformaldehyde gave E, which was deprotected by hydrogenation
and
alkylated with benzyl bromoacetate to give G, which was finally hydrogenated
to H. Rink
amide resin functionalized with the octapeptide Gln-Trp-Ala-Val-Gly-His-Leu-
Met-NHZ
(BBN[7-14]) (A) was sequentially reacted with Fmoc-4-aminobenzoic acid, Fmoc-
glycine
and H. After cleavage and deprotection with Reagent B the crude was purified
by
preparative HPLC to give L237. Overall yield 0.21 %.
A. 7-Formyl-1,4,7,10-tetraazacyclododecane-1-carboxylic
acid phenylmethyl ester dihydrochloride, B (FIG. 10A)
[00291] 1-Formyl-1,4,7,10-tetraazacyclododecane A (14 g; 69.9 mmol) was
dissolved
in H20 (100 mL) and 12 N HGl (11 mL) was added until pH 3 then 1,4-
dioxane (220 mL) was added. A solution of benzyl chloroformate (13.8 g; 77
mmol) in 1,4-dioxane (15 mL) was slowly added dropwise in 3.5 h, constantly
maintaining the reaction mixture at pH 3 by continuous addition of 2 N NaOH
(68.4 mL) with a pHstat apparatus. At the end of the addition the reaction was
stirred for 1 h then washed with n-hexane (4 x 100 mL) and'Pr20 (4 x 100
mL). The aqueous phase was brought to pH 13 by addition of 10 N NaOH
(6.1 mL) and extracted with CHC13 (4 x 100 mL). The organic phase was
washed with brine (100 mL), dried (Na2S04), filtered and evaporated. The
oily residue was dissolved in acetone (200 mL) and 6 N HCl (26 mL) was
added. The solid precipitated was filtered, washed with acetone (2 x 50 mL)
and dried under vacuum to give compound B (23.6 g; 58 mmol) as a white
crystalline solid. Yield 83%.
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B. 4-(Phenylmethoxy)carbonyl-1,4,7,10-tetraazacyclododecane-
1,7-diacetic acid bis(1,1-dimethylethyl) ester D (FIG 10A)
[00292] A solution of B (14.4 g; 35.3 mmol) in H20 (450 mL) and 1 N NaOH (74
mL;
74 mmol) was stirred for 20 min then extracted with CHCl3 (4 x 200 mL).
The organic layer was evaporated to obtain an oily residue (12.3 g) which was
dissolved in CH3CN (180 mL) and N ethyldiisopropylamine (DIEA) (15 mL;
88.25 mmol). A solution of t-butyl bromoacetate (16.8 g; 86.1 mmol) in
CH3CN (15 mL) was added dropwise to the previous solution in 2.5 h. After
20 h at room temperature the solvent was evaporated and the oily residue was
dissolved in CHCl3 (150 mL) and washed with H20 (5 x 100 mL). The
organic layer was dried (Na2S04), filtered and evaporated to dryness to give C
as a yellow oil. Crude C (22 g) was dissolved in EtOH (250 mL),
NH20H~HC1 (2.93g; 42.2 mmol) was added and the solution heated to reflux.
After 48 h the solvent was evaporated and the residue dissolved in CHZCh
(250 mL), washed with HBO (3 x 250 mL) then with brine (3 x 250 mL). The
organic layer was dried (Na2S04), filtered and evaporated. The oily residue
(18.85 g) was purified by flash chromatography. The fractions containing the
product were collected and evaporated to obtain a glassy white solid (17.62 g)
which was dissolved in H20 (600 mL) and 1 N NaOH (90 mL; 90 mmol) and
extracted with CHC13 (3 x 250 ml). The organic layer was dried (Na2S04) and
evaporated to dryness to give D (16.6 g; 31 mmol) as an oil. Yield 88%.
C. 4-(Phenylmethoxy)carbonyl-10-[[bis( 1,1-
dimethylethoxy)phosphinyl]methyl)-1,4,7,10-
tetraazacyclododecane-1,7-diacetic acid bis(1,1-
dimethylethyl) ester. E (FIG. 10A)
[00293] A mixture of Compound D (13.87 g; 26 mmol), P(OtBu)3 (7.6 g; 28.6
mmol)
(10) and paraformaldeyde (0.9 g; 30 mmol) was heated at 60 °C. After 16
h
more P(OtBu)3 (1 g; 3.76 mmol) and paraformaldeyde (0.1 g; 3.33 mmol)
were added. The reaction was heated at 60 °C for another 20 h then at
80 °C
for 8 h under vacuum to eliminate the volatile impurities. The crude was
purified by flash chromatography to give E (9.33 g; 8 mmol) as an oil. Yield
31 %.
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D. 7-[ [Bis( 1,1-dimethylethoxy)phosphinyl]methyl]-1,4,7,10-
tetraazacyclododecane-1,4,10-triacetic acid 1-phenylmethyl
4,10-bis( 1,1-dimeth l~yl) ester G (FIG 10A)
[00294] To the solution of E (6.5 g; 5.53 mmol) in GH3OH (160 mL) 5% Pd/C (1
g;
0.52 mmol) was added and the mixture was stirred under hydrogen
atmosphere at room temperature. After 4 h (consumed HZ 165 mL;6.7 mmol)
the mixture was filtered through a Millipore~ filter (FT 0.45 ~.m) and the
solution evaporated under reduced pressure. The crude (5.9 g) was purified by
flash chromatography to give F (4.2 g) as an oil. Benzyl bromoacetate (1.9 g;
8.3 mmol) dissolved in CH3CN (8 mL) was added dropwise in 1 h to a
solution of F (4.2 g) in CH3CN (40 mL) and DIEA (1.5 mL; 8.72 mmol).
After 36 h at room temperature the solvent was evaporated and the residue
(5.76 g) dissolved in CHC13 (100 mL), washed with H20 (2 x 100 mL) then
with brine (2 x 70 mL). The organic layer was dried (Na2S04), filtered and
evaporated. The crude (S.5 g) was purified twice by flash chromatography,
the fractions were collected and evaporated to dryness to afford G (1.12 g;
1.48 mmol) as an oil. Yield 27%.
E. 7-[ [Bis( 1,1-dimethylethoxy)phosphinyl]methyl]-1,4,7,10-
tetraazacyclododecane-1,4,10-triacetic acid 4,10-bis(1,1-dimethylethyl)
ester, H (FIG. 10A)
[00295] 5% Pd/C (0.2 g; 0.087 mmol) was added to a solution of G (1.12 g; 1.48
mmol) in CH30H (27 mL) and the mixture was stirred under hydrogen
atmosphere at room temperature. After 2 h (consumed H2 35 mL; 1.43 mmol)
the mixture was ftltered through a Millipore~ filter (FT 0.45 Vim) and the
solution evaporated to dryness to give H (0.94 g; I .41 mmol) as a pale yellow
oil. Yield 97%.
F. N [4-[[[[[4,10-Bis(carboxymethyl)-7-(dihydroxyphosphinyl)methyl-
1,4,7,10-tetraazacyclododec-1-yl] acetyl] amino] acetyl] amino]benzoyl]-L-
glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucil-L-
methioninamide, L237 (FIG. 10B)
[00296] Resin A (330 mg; 0.20 mmol) (17) was shaken in a solid phase peptide
synthesis vessel with 50% morpholine in DMA (5 mL) for 10 min, the
solution emptied and fresh 50 % morpholine in DMA (5 mL) was added. The
suspension was stirred for another 20 min then the solution was emptied and
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the resin washed with DMA (5 x 5 mL). Fmoc-4-aminobenzoic acid (290 mg;
0.80 mmol), HOBt (120 mg; 0.80 mmol), DIC (130 ~L; 0.80 mmol) and DMA
(5 mL) were added to the resin, the mixture shaken for 3 h at room
temperature, emptied and the resin washed with DMA (5 x 5 mL). The resin
was then shaken with 50% morpholine in DMA (5 mL) for I 0 min, the
solution emptied, fresh 50% morpholine in DMA (5 mL) was added and the
mixture shaken for another 20 min. The solution was emptied and the resin
washed with DMA (5 x 5 mL). Fmoc-glycine (240 mg; 0.8 mmol), HATU
(310 mg; 0.8 mmol) and DIEA (260 ~.L; 1.6 mmol) were stirred for 15 min in
DMA (5 mL) then the solution was added to the resin, the mixture shaken for
2 h at room temperature, emptied and the resin washed with DMA (5 x 5 mL).
The resin was then shaken with 50% morpholine in DMA (5 mL) for 10 min,
the solution emptied, fresh 50% morpholine in DMA (5 mL) was added and
the mixture shaken for another 20 min. The solution was emptied and the
resin washed with DMA (5 x 5 mL). H (532 mg; 0.80 mmol), HOBt (120 mg;
0.80 mmol), DIC (130 ~.L; 0.80 mmol), and DIEA (260 ~.L; 1.6 mmol) and
DMA (5 mL) were added to the resin. The mixture was shaken in a flask for
40 h at room temperature, emptied and the resin washed with DMA (5 x 5
mL), CH2C12 (5 x 5 mL) and vacuum dried. The resin was shaken in a flask
with Reagent B (25 mL) for 4 h. The resin was filtered and the solution was
evaporated under reduced pressure to afford an oily crude that after treatment
with Et20 (20 mL) gave a precipitate. The precipitate was collected by
centrifugation and washed with Et20 (3 x 20 mL) to give a solid (90 mg)
which was analysed by HPLC. An amount of crude (50 mg) was purified by
preparative HPLC. The fractions containing the product were lyophilised to
give L237 (6 mg; 3.9 x 10-3 mmol) as a white solid. Yield 3.5%.
EXAMPLE XI - Figures 11A-B
Synthesis of L238 and L239
[00297] Summary: The products were obtained in several steps starting from the
octapeptide Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2 (BBN[7-14]) (A) on the Rink
amide
resin. After cleavage and deprotection with Reagent B the crude was purified
by preparative
HPLC to give L238 and L239. Overall yields: 14 and 9%, respectively.
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101
A. N,N Dimethylglycyl-L-Beryl-[S-[(acetylamino)methyl]]-L-cysteinyl-
glycyl-4-aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-
glycyl-L-histidyl-L-leucyl-L-methioninamide, L238 (FIG.11A)
[00298] Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide
synthesis vessel
with 50% morpholine in DMA (7 mL) for 10 min, the solution emptied and
fresh 50% morpholine in DMA (7 mL) was added. The suspension was stirred
for another 20 min then the solution was emptied and the resin washed with
DMA (5 x 7 mL). Fmoc-4-aminobenzoic acid (0.43 g; 1.2 mmol), HOBt (0.18
g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol) and DMA (7 mL) were added to the
resin, the mixture shaken for 3 h at room temperature, emptied and the resin
washed with DMA (5 x 7 mL). The resin was then shaken with 50%
morpholine in DMA (7 mL) for 10 min, the solution emptied, fresh 50%
morpholine in DMA (7 mL) was added and the mixture shaken for another 20
min. The solution was emptied and the resin washed with DMA (5 x 7 mL).
Fmoc-glycine (0.36 g; 1.2 mmol), HATU (0.46 g; 1.2 mmol) and N
ethyldiisopropylamine (0.40 mL; 2.4 mmol) were stirred for 15 min in DMA
(7 mL) then the solution was added to the resin, the mixture shaken for 2 h at
room temperature, emptied and the resin washed with DMA (5 x 7 mL). The
resin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the
solution emptied, fresh 50% morpholine in DMA (7 mL) was added and the
mixture shaken for another 20 min. The solution was emptied and the resin
washed with DMA (5 x 7 mL). N a-Fmoc-S-acetamidomethyl-L-cysteine
(0.50 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol) and
DMA (7 mL) were added to the resin, the mixture shaken for 3 h at room
temperature, emptied and the resin washed with DMA (5 x 7 mL). The resin
was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the
solution emptied, fresh 50% morpholine in DMA (7 mL) was added and the
mixture shaken for another 20 min. The solution was emptied and the resin
washed with DMA (5 x 7 mL). N a-Fmoc-O-t-butyl-L-serine (0.46 g; 1.2
mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), and DMA (7
mL) were added to the resin, the mixture was shaken for 3 h at room
temperature, emptied and the resin washed with DMA (5 x 7 mL). The resin
was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the
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solution emptied, fresh 50% morpholine in DMA (7 mL) was added and the
mixture shaken for another 20 min.
[00299] The solution was emptied and the resin washed with DMA (5 x 7 mL). N,N
Dimethylglycine (0.12 g; 1.2 mmol), HATU (0.46 g; 1.2 mmol) and N
ethyldiisopropylamine (0.40 mL; 2.4 mmol) were stirred for 15 min in DMA
(7 mL) then the solution was added to the resin. The mixture was shaken for 2
h at room temperature, emptied and the resin washed with DMA (5 x 7 mL),
CH2Cl2 (5 x 7 mL) and vacuum dried. The resin was shaken in a flask with
Reagent B (25 mL) for 4.5 h. The resin was filtered and the solution was
evaporated under reduced pressure to afford an oily crude that after treatment
with Et2O (20 mL) gave a precipitate. The precipitate was collected by
centrifugation and washed with Et20 (3 x 20 mL) to give a solid (169 mg)
which was analysed by HPLC. An amount of crude (60 mg) was purified by
preparative HPLC. The fractions containing the product were lyophilised to
give L238 (22.0 mg; 0.015 mmol) as a white solid. Yield 14%.
B. N,N Dimethylglycyl-L-Beryl-[S-[(acetylamino)methyl]]-L-cysteinyl-
glycyl-(313,513,7a, l 2a)-3-amino-7,12-dihydroxy-24-oxocholan-24-yl-L-
glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-Ieucyl-L-
methioninamide. L239 (FIG. llBl
[00300] Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide
synthesis vessel
with 50% morpholine in DMA (7 mL) for 10 min, the solution emptied and
fresh 50% morpholine in DMA (7 mL) was added. The suspension was stirred
for another 20 min then the solution was emptied and the resin washed with
DMA (5 x 7 mL). (313,S13,7a,12a)-3-[[(9H-Fluoren-9-
ylmethoxy)amino]acetyl]amino-7,12-dihydroxycholan-24-oic acid B (0.82 g;
1.2 mmol) (7), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol) and DMA
(7 mL) were added to the resin, the mixture shaken for 24 h at room
temperature, emptied and the resin washed with DMA (5 x 7 mL). The resin
was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the
solution emptied, fresh 50% morpholine in DMA (7 mL) was added and the
mixture shaken for another 20 min. The solution was emptied and the resin
washed with,DMA (5 x 7 mL). N a-Fmoc-S-acetamidomethyl-L-cysteine
(0.50 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol) and
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DMA (7 mL) were added to the resin, the mixture was shaken for 3 h at room
temperature, emptied and the resin washed with DMA (5 x 7 mL). The resin
was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the
solution emptied, fresh 50% morpholine in DMA (7 mL) was added and the
mixture shaken for another 20 min. The solution was emptied and the resin
washed with DMA (5 x 7 mL). N a-Fmoc-O-t-butyl-L-serine (0.46 g; 1.2
mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), and DMA (7
mL) were added to the resin, the mixture was shaken for 3 h at room
temperature, emptied and the resin washed with DMA (5 x 7 mL). The resin
was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the
solution emptied, fresh 50% morpholine in DMA (7 mL) was added and the
mixture shaken for another 20 min. The solution was emptied and the resin
washed with DMA (5 x 7 mL). N,N Dimethylglycine (0.12 g; 1.2 mmol),
HATU (0.46 g; 1.2 mmol) and N ethyldiisopropylamine (0.40 mL; 2.4 mmol)
were stirred for 15 min in DMA (7 mL) then the solution was added to the
resin.
[00301] Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide
synthesis vessel
with 50% morpholine in DMA (7 mL) for 10 min, the solution emptied and
fresh 50% morpholine in DMA (7 mL) was added. The suspension was stirred
for another 20 min then the solution was emptied and the resin washed with
DMA (5 x 7 mL). (313,513,7a,12a)-3-[[(9H-Fluoren-9-
ylmethoxy)amino]acetyl]amino-7,12-dihydroxycholan-24-oic acid B (0.82 g;
1.2 mmol) HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2 mrnol) and DMA (7
mL) were added to the resin, the mixture shaken for 24 h at room temperature,
emptied and the resin washed with DMA (5 x 7 mL). The resin was then
shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution
emptied, fresh 50% morpholine in DMA (7 mL) was added and the mixture
shaken for another 20 min. The solution was emptied and the resin washed
with DMA (5 x 7 mL). N a-Fmoc-S-acetamidomethyl-L-cysteine (0.50 g; 1.2
mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol) and DMA (7 mL)
were added to the resin, the mixture was shaken for 3 h at room temperature,
emptied and the resin washed with DMA (5 x 7 mL). The resin was then
shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution
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emptied, fresh 50% morpholine in DMA (7 mL) was added and the mixture
shaken for another 20 min. The solution was emptied and the resin washed
with DMA (5 x 7 mL). N a-Fmoc-O-t-butyl-L-serine (0.46 g; 1.2 mmol),
HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), and DMA (7 mL) were
added to the resin, the mixture was shaken for 3 h at room temperature,
emptied and the resin washed with DMA (5 x 7 rnL). The resin was then
shaken with 50% morpholine in DMA (7 mL) for 10 min, the solution
emptied, fresh 50% morpholine in DMA (7 mL) was added and the mixture
shaken for another 20 min. The solution was emptied and the resin washed
with DMA (5 x 7 mL). N,N Dimethylglycine (0.12 g; 1.2 mmol), HATU
(0.46 g; 1.2 mmol) and N ethyldiisopropylamine (0.40 mL; 2.4 mmol) were
stirred for 15 min in DMA (7 mL) then the solution was added to the resin.
EXAMPLE XII - Figures 12A-F
Synthesis of L240, L241, L242
[00302] Summary: The products were obtained in several steps starting from the
octapeptide Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2 (BBN[7-14]) (A) on the Rink
amide
resin. After cleavage and deprotection with Reagent B the crudes were purified
by
preparative HPLC to give L240, L241, and L242. Overall yields: 7.4, 3.2, 1.3%
respectively.
A. 4-[[[(9H Fluoren-9-
ylmethoxy)carbonyl]amino]acetyl]amino-3-methoxybenzoic
acid A (FIG. 12A)
[00303] A solution of 4-amino-3-methoxybenzoic acid (1.0 g; 5.9 mmol); and N-
ethyldiisopropylamine (1.02 mL 5.9 mmol) in THF (20 mL) was added
dropwise to a solution of Fmoc-glycylchloride (1.88 g; 5.9 mmol) in CHaCl2
/THF 1:1 (20 rnL) and stirred at room temperature under N2. After 3 h the
solvent was evaporated under vacuum. The residue was taken up with 0.5 N
HCl (50 mL), was stirred for 1 h at 0 °C then filtered and dried.
The white
solid was suspended in MeOH (30 mL) and stirred for I h, then was filtered
and suspended in a solution of CHC13/hexane 1:4 (75 mL). The suspension
was filtered to give compound A as a with solid (1.02 g; 2.28 mmol). Yield
39%.
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B. N [4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10tetraazacyclododec-1-yl]
acetyl]glycylJaminoJ-3-methoxybenzoyl)-L-glutaminyl-L-tryptophyl-1-
alanyl-L-valyl-~Iycvl-L-histidyl-L-leucyl L methioninamide L240
[00304] Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide
synthesis vessel
with 50% morpholine, in DMA (7 mL) for 10 min, the solution emptied and
fresh SO% morpholine in DMA (7 mL) was added. The suspension was stirred
for another 20 min then the solution was emptied and the resin washed with
DMA (5 x 7 mL). 4-[[[ (9H-Fluoren-9-ylmethoxy)carbonylJ amino] acetyl]
amino-3-methoxybenzoic acid, A (0.50 g; 1.2 mmol), HOBt (0.18 g; 1.2
mmol), DIC (0.19 mL; 1.2 mmol) and DMA (7 mL) were added to the resin,
the mixture shaken for 5 h at room temperature, emptied and the resin washed
with DMA (5 x 7 mL). The resin was then shaken with 50% morpholine in
DMA (7 mL) for 10 min, the solution emptied, fresh 50% morpholine in DMA
(7 mL) was added and the mixture shaken for another 20 min. The solution
was emptied and the resin washed with DMA (5 x 7 mL). 1,4,7,10-
Tetraazacyclododecane-1,4,7,10-tetraacetic acid tris(l,l-dimethylethyl) ester
adduct with NaCI (0.79 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19
mL: 1.2 mmol}, N ethyldiisopropylamine (0.40 mL; 2.4 mmol) and DMA (7
mL} were added to the resin. The mixture was shaken for 24 h at room
temperature, emptied and the resin washed with DMA (5 x 7 mL), CH2C12 (5 x
7 mL) and vacuum dried. The resin was shaken in a flask with Reagent B (25
mL) for 4.5 h. The resin was filtered and the solution was evaporated under
reduced pressure to afford an oil crude that after treatment with Et2O (20 mL)
gave a precipitate. The precipitate was collected by centrifugation and washed
with EtzO (5 x 20 mL) to give a solid (152 mg) which was analysed by HPLC.
An amount of crude (52 mg) was purified by preparative HPLC. The fractions
containing the product were lyophilised to give L240 (12.0 mg; 7.8 x 10-3
mmol) as a white solid. Yield 7.4%.
C. 4-amino-3-chlorobenzoic acid C (FIG 12B)
[00305) 1 N NaOH (1 I mL; 11 mmol) was added to a solution ofmethyl 4-amino-3-
chlorobenzoate (2 g; 10.8 mmol) in MeOH (20 mL) at 45 °C. The reaction
mixture was stirred for 5 h at 45 °C and overnight at room temperature.
More
1N NaOH was added (5 mL; 5 mmol) and the reaction was stirred at 45 °C
for
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2 h. After concentration of solvent was added 1N HCl (16 ml). The solid
precipitate was filtered and dried to give 4-amino-3-chlorobenzoic acid, C, as
a with solid (I,75 g; 10.2 mmol). Yield 94.6%.
D. 4-[[[(9H Fluoren-9-ylmethoxy)carbonyl]amino]acetyl]amino-
3-chlorobenzoic acid, D (FIG. 12B1
[00306] A solution of 4-amino-3-chlorobenzoic acid (1.5 g; 8.75 mmol) and N-
ethyldiisopropylamine (1.46 mL 8.75 mmol) in THF (50 mL) was added
dropwise to a solution of Fmoc-glycylchloride (2.76 g; 8.75 mmol) in CH2Cl2
/THF 1:1 (30 mL) and stirred at room temperature under N2. After 3 h the
solvent was evaporated under vacuum. The residue was taken up with 0.5N
HCl (50 mL), filtered and dried.
The white solid was suspended in MeOH (30 mL) and stirred for 1 h, then was
filtered and dried to give 4-[[[(9H-fluoren-9-
ylmethoxy)carbonyl]amino]acetyl]amino-3-chlorobenzoic acid (2.95 g; 6.5
mmol). Yield 75%.
E. N [4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,lOtetraazacyclododec-1-yI]
acetyl] glycyl] amino] 3-chlorob enzoyl] L-glutaminyl-L-tryptophyl-1-
alanyl-L-valy~l~yl-L-histidyl-L-leucyl-L-methioninamide L241
(FIG. 12E)
[00307] Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide
synthesis vessel
with 50% morpholine in DMA (7 mL) for 10 min, the solution emptied and
fresh 50% morpholine in DMA (7 mL) was added. The suspension was stirred
for another 20 min then the solution was emptied and the resin washed with
DMA (5 x 7 mL). 4-[[[(9H Fluoren-9-
ylmethoxy)carbonyl]amino]acetyl]amino-3-chlorobenzoic acid, D (0.54 g; 1.2
mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol) and DMA (7 mL)
were added to the resin, the mixture shaken for 5 h at room temperature,
emptied and the resin washed with DMA (5 x 7 mL).
[00308] The resin was then shaken with 50% morpholine in DMA (7 mL) for 10
min,
the solution emptied, fresh 50% morpholine in DMA (7 mL) was added and
the mixture shaken for another 20 min. The solution was emptied and the
resin washed with DMA (5 x 7 mL). 1,4,7,10-Tetraazacyclododecane-
1,4,7,10-tetraacetic acid tris(l,l-dimethylethyl) ester adduct with NaCl (0.79
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g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), N
ethyldiisopropylamine (0.40 mL; 2.4 mmol) and DMA (7 mL) were added to
the resin. The mixture was shaken for 40 h at room temperature, emptied and
the resin washed with DMA (5 x 7 mL), CHZCl2 (5 x 7 mL) and vacuum dried.
The resin was shaken in a flask with Reagent B (25 mL) for 4.5 h. The resin
was filtered and the solution was evaporated under reduced pressure to afford
an oil crude that after treatment with Et20 (20 mL) gave a precipitate. The
precipitate was collected by centrifugation and washed with Et20 (5 x 20 mL)
to give a solid (151 mg) which was analysed by HPLC. An amount of crude
(56 mg) was purified by preparative HPLC. The fractions containing the
product were lyophilised to give L241 (5.6 mg; 3.6 x 10-3 mmol) as a white
solid. Yield 3.2%.
F. 4-[[[(9H Fluoren-9-
ylmethoxy)carbonyl]amino]acetyl]amino-3-methylbenzoic
acid, E (FIG. 12C)
[00309] A solution of 4-amino-3-methylbenzoic acid (0.81 g; 5.35 mmol) and N
ethyldiisopropylamine (0.9 mL 5.35 mmol) in THF (30 mL) was added
dropwise to a solution of Fmoc-glycylchloride (1.69 g; 5.35 mmol) in CH2C12
/THF 1:1 (20 mL) and stirred at room temperature under N2. After 3 h the
solvent was evaporated under vacuum. The residue was taken up with HCl
0.5 N (50 mL)and was stirred for 3 h at 0°C. then was filtered and
dried. The
white solid was suspended in MeOH (50 mL) and stirred for 1 h, then filtered
and dried to give Compound E (1.69 g; 3.9 mmol). Yield 73%.
G. N-[4-[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]
2 5 acetyl] glycyl] amino] 3-methylb enzoyl] L-glutaminyl-L-tryptophyl-L-
alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide L242
(FIG. 12F1
[00310] Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide
synthesis vessel
with 50% morpholine in DMA (7 mL) for 10 min, the solution emptied and
fresh 50% morpholine in DMA (7 mL) was added. The suspension was stirred
for another 20 min then the solution was emptied and the resin washed with
DMA (5 x 7 mL). 4-[[(9H-Fluoren-9-ylmethoxy)amino]acetyl]amino-3-
methylbenzoic acid, E (0.52 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC
(0.19 mL; 1.2 mmol) and DMA (7 mL) were added to the resin, the mixture
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shaken for 5 h at room temperature, emptied and the resin washed with DMA
(5 x 7 mL). The resin was then shaken with 50% morpholine in DMA (7 mL)
for 10 min, the solution emptied, fresh 50% morpholine in DMA (7 mL) was
added and the mixture shaken for another 20 min. The solution was emptied
and the resin washed with DMA (5 x 7 mL).1,4,7,10-Tetraazacyclododecane-
1,4,7,10-tetraacetic acid tris(1,1-dimethylethyl) ester adduct with NaCl (0.76
g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol), N
ethyldiisopropylamine (0.40 mL; 2.4 mmol) and DMA (7 mL) were added to
the resin.
[00311] The mixture was shaken for 40 h at room temperature, emptied and the
resin
washed with DMA (5 x 7 mL), CHZC12 (5 x 7 mL) and vacuum dried. The
resin was shaken in a flask with Reagent B (25 mL) for 4.5 h. The resin was
filtered and the solution was evaporated under reduced pressure to afford an
oil crude that after treatment with Et20 (20 mL) gave a precipitate. The
precipitate was collected by centrifugation and washed with EtZO (5 x 20 mL)
to give a solid (134 mg) which was analysed by HPLC. An amount of crude
(103 mg) was purified by preparative HPLC. The fractions containing the
product were lyophilised to give L242 (4.5 mg; 2.9 x 10-3 mmol) as a white
solid. Yield 1.3%.
EXAMPLE XIII - Figures 13A-C
Synthesis of L244
[00312] Summary: The product was obtained in several steps starting from the
octapeptide Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2 (BBN[7-14]) on the Rink amide
resin
(A). The final coupling step with DOTA tri-t-butyl ester was done in solution
phase after
cleavage and deprotection with Reagent B of Linker-BBN [7-14]. The crude was
purified by
preparative HPLC to give L244. Overall yield: 0.4%.
A. N.N'-(Iminodi-2,1-ethanedi~)bis[2 2 2-trifluoroacetamide~~FIG
13A
[00313] Trifluoroacetic acid ethyl ester (50 g; 0.35 mol) was dropped into a
solution of
diethylenetriamine (18 g; 0.175 mol) in THF (180 mL) at 0°C in 1 h.
After 20
h at room temperature, the mixture was evaporated to an oily residue (54 g). T
he oil was crystallized from Et20 (50 mL), filtered, washed with cooled Et2O
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(2 x 30 mL) and dried to obtain A as a white solid (46 g; 0.156 mol). Yield
89%.
B. 4-[N,N'-Bis[2-(trifluoroacetyl)aminoethyl]amino]-4-oxobutanoic
acid, B (FIG. 13A)
[00314] Succinic anhydride (0.34 g; 3.4 mmol) was added in a solution of A (1
g; 3.4
mmol) in THF (5 mL) at room temperature. After 28 h the crude was
concentrated to residue (1.59 g), washed with EtOAc (2 x 10 mL) and 1 N
HCl (2 x 15 mL). The organic layer was dried on Na2S04, filtered and
evaporated to give an oily residue (1.3 g) that was purified by flash
chromatography (5) to afford B as an oil (0.85 g; 2.15 mmol). Yield 63%.
C. 4-[N,N'-Bis[2-[(9-H fluoren-9-
ylmethoxy)carbonyl]aminoethyl]amino]-4-oxobutanoic acid, D (FIG.
[00315] Succinic anhydride (2 g; 20 mmol) was added in a solution of A (5 g;
16.94
mmol) in THF (25 mL) at room temperature. After 28 h the crude was
concentrated to residue (7 g), washed in ethyl acetate (100 mL) and in 1 N
HCI (2 x 50 mL). The organic layer was dried on Na2S04, filtered and
evaporated to give crude B as an oily residue (6.53 g). 2 N NaOH (25 mL) was
added to suspension of crude B (5 g) in EtOH (35 mL) obtaining a complete
solution after 1 h at room temperature. After 20 h the solvent was evaporated
to obtain C as an oil (8.48 g). A solution of 9-fluorenylmethyl chloroformate
(6.54 g, 25.3 mmol) in 1,4-dioxane (30 mL), was dropped in the solution of C
in 10% aq. Na2C03 (30 mL) in 1 h at 0°C. After 20 h at r.t. a
gelatinous
suspension was obtained and filtered to give a white solid (3.5 g) and a
yellow
solution. The solution was evaporated and the remaining aqueous solution
was diluted in H20 (150 mL) and extracted with EtOAc (70 mL). Fresh
EtOAc (200 mL) was added to aqueous phase, obtaining a suspension which
was cooled to 0°C and acidified to pH 2 with conc. HCI. The organic
layer
was washed with H20 (5 x 200 mL) until neutral pH, then dried to give a
glassy solid (6.16 g). The compound was suspended in boiling n-Hexane (60
mL) for 1 h, filtered to give D as a white solid (5.53 g, 8.54 mmol). Overall
yield 50%.
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D. N [4-[[4-[Bis[2-[[[4,7,10-tris(carboxymethyl)-1,4,7,10-
tetraazacyclododec-1-ylJacetylJamino]ethyl]amino-1,4-
dioxobutyl]amino]benzoyl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-
valyl-g-l~cyl-L-histidyl-L-leu~l-L-methioninamide. L244 FIG. 13B)
[00316] Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide
synthesis vessel
with 50% morpholine in DMA (7 mL) for 10 min, the solution emptied and
fresh 50% morpholine in DMA (7 mL) was added. The suspension was stirred
for another 20 min then the solution was emptied and the resin washed with
DMA (5 x 7 mL). 4-[N,N'-Bis[2-[(9-H-fluoren-9-yl methoxy) carbonyl]
aminoethylJ amino]-4-oxo butanoic acid (777.3 mg; 1.2 mmol), HOBt (184
mg; 1.2 mmol), DIC (187 ~,L; 1.2 mmol) and DMA (7 mL) were added to the
resin, the mixture shaken for 40 h at room temperature, emptied and the resin
washed with DMA (5 x 7 mL). The resin was then shaken with 50%
morpholine in DMA (7 mL) for 10 min, the solution emptied, fresh 50%
morpholine in DMA (7 mL) was added and the mixture shaken for 20 min.
The solution was emptied and the resin washed with DMA (2 x 7 mL) and
with CH2Cl2 (5 x 7 mL) then it was shaken in a flask with Reagent B (25 mL)
for 4.5 h. The resin was filtered and the solution was evaporated under
reduced pressure to afford an oily crude that after treatment with Et20 (20
mL)
gave a precipitate. The precipitate was collected by centrifugation and
washed with Et20 (5 x 20 mL) to give F as a white solid (140 mg). DOTA tri-
t-butyl ester (112 mg; 0.178 mmol) HATU (70 mg; 0.178 mmol) and DIEA
(60 ~.L; 0.356 mmol) were added to a solution of F (50 mg; 0.0445 mmol) in
DMA (3 mL) and CH2Cl2 (2 mL) and stirred for 24 h at room temperature.
The crude was evaporated to reduced volume (1 rnL) and shaken with Reagent
B (25 mL) for 4.5 h. After evaporation of the solvent, the residue was treated
with EtaO (20 mL) to give a precipitate. The precipitate was collected by
centrifugation and washed with Et20 (5 x 20 mL) to afford a beige solid (132
mg) that was analyzed by HPLC. An amount of crude (100 mg) was purified
by preparative HPLC. The fractions containing the product were lyophilized
to give L244 (FIG. 13C) (3.5 mg; 1.84 x 10-3 mmol) as a white solid. Yield
0.8 %.
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General Experimentals for Examples XIV-Example XLII
L201-L228
A. Manual Cou~lyn~s
[00317] 6.0 equivalents of the appropriately protected amino acid was treated
with 6.0
equivalents each of HOBt and DIC and activated outside the reaction vessel.
This activated carboxylic acid in NMP was then transferred to the resin
containing the amine and the reaction was carried out for 4-6 h and then the
resin was drained and washed.
B. Special coupling of Fmoc-Gly-OH to 4-aminobenzoic acid and
aminobiphenylcarboxylic acid amides'
[00318) Fmoc-Gly-OH (10.0 equiv.) was treated with HATU (10.0 equiv.) and DIEA
(20.0 equiv.) in NMP (10 mL of NMP was used for one gram of the amino
acid by weight) and the solution was stirred for 10-15 min at RT before
transfernng to the vessel containing the amine loaded resin. The volume of
the solution was made to 15.0 ml for every gram of the resin. The coupling
was continued for 20h at RT and the resin was drained of all the reactants.
This procedure was repeated one more time and then washed with NMP
before moving on to the next step.
C. Preparation of D03A monoamide~
[00319) 8.0 equivalents of DOTA mono acid was dissolved in NMP and treated
with
8.0 equivalents of HBTU and 16.0 equivalents of DIEA. This solution was
stirred for 15 min at RT and then transferred to the amine on the resin and
the
coupling was continued for 24h at RT. The resin was then drained, washed
and then the peptide was cleaved and purified.
D. Cleavage of the crude peptides from the resin and burification~
[00320] The resin was suspended in Reagent B (15.0 ml/g) and shaken for 4h at
RT.
The resin was then drained and washed with 2 x 5 mL of Reagent B again and
combined with the previous filtrate. The filtrate was then concentrated under
reduced pressure to a pastelliquid at RT and triturated with 25.0 mL of
anhydrous ether (for every gram of the resin used). The suspension was then
centrifuged and the ether layer was decanted. This procedure was repeated
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two more times and the colorless precipitate after ether wash was purified by
preparative HPLC.
Example XIV - Figure 21
Synthesis of L201
[00321 ] 0.5 g of the Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-M-Resin (0.4 mmol/g,
O.Sg,
0.2 mmol) (Resin A) was used. The rest of the amino acid units were added as
described in
the general procedure to prepare ( 1 R)-I -(Bis {2-
[bis(carboxymethyl)amino]ethyl}amino)propane-3-carboxylic acid-1-carboxyl-
glycyl-4-
aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-
leucyl-L-
methioninamide (L201), Yield: 17.0 mg (5.4%)
Examine XV - Figures 22A and 22B
Synthesis of L202
A. 4-Fmoc-hydrazinobenzoic acid (Fig 2~A~
[00322] A suspension of 4-hydrazinobenzoic acid (5.0 g, 32.9 mmol) in water
(100 ml)
was treated with cesium carbonate (21.5 g, 66.0 mmol). Fmoc-CI (9.1 g, 35.0
mmol) in THF (25 mL) was added dropwise to the above solution with stirring
over a period of 1h. The solution wasrstirred for 4h more after the addition
and the reaction mixture was concentrated to about 75 mL and extracted with
ether (2 x 100 mL). The ether layer was discarded and the aqueous layer was
acidified with 2N HCl. The separated solid was filtered, washed with water (5
x 100 mL) and then recrystallized from acetonitrile to yield the product
(compound B) as a colorless solid. Yield: 11.0 g (89%). 1H NMR (DMSO-db)
d 4.5 (m, 1 H, Ar-CH -CH), 4.45 (m, 2H, Ar-CH ), 6.6 (bs, 1 H, Ar-H), 7.4 -
7.9 (m, 9, Ar-H and Ar-CH ), 8.3 (s, 2H, Ar-H), 9.6 (s, 2H, Ar-H). M. S. -
m/z 373.2 [M-H].
[00323] 0.5 g of the Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-M-Resin (0.4 mmol/g,
O.Sg,
0.2 mmol) (Resin A) was used. The amino acid units were added as described
in the general procedure, including Compound B to prepare N [(313,513,12a)-3-
[ [[[ [4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-
3 0 y1] acetyl] amino] acetyl] amino]-4-hydrazinob enzoyl-L-glutaminyl-L-
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tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninarnide
(L202) (Fig. 22B), Yield: 25.0 mg (8.3%).
Example XVI - Figures 23A and 23B
Synthesis of L203
A. Preparation of 4-Boc-aminobenzyl benzoate Compound B FIG. 23A~
[00324] A suspension of 4-boc-aminobenzoic acid (0.95 g, 4.0 mmol) in dry
acetonitrile (10.0 mL) was treated with powdered cesium carbonate (1.3 g, 4.0
mmol) and stirred vigorously under nitrogen. Benzyl bromide (0.75 g, 4.4
mmol) was added and the reaction mixture was refluxed for 20h under
nitrogen. The reaction mixture was then poured into ice cold water (200 mL)
and the solid separated was filtered and washed with water (5 x 50 mL). The
crude material was then recrystallized from aqueous methanol to yield the
product as a colorless solid (Compound B). Yield: 0.8 g (61 %). 'H NMR
(CDC13): d 1.5 (s, 9H, Tertiary methyls), 5.4 (s, 2H, Ar-CH ), 7.4 (m, 7H, Ar-
H) and 8.0 (m, 2H, Ar-H). M. S. -m/z 326.1 [M+H].
B. 4-Aminobenzyl benzoate Compound C (FIG. 23A~
[00325] 4-Boc-aminobenzyl benzoate (0.8 g, 2.5 mmol) was dissolved in DCM (20
mL) containing TFA (25% by volume) and stirred for 2h at RT. The reaction
mixture was poured into 100.0 g of crushed ice and neutralized with saturated
sodium bicarbonate solution until the pH reached about 8.5. The organic layer
was separated and the aqueous layer was extracted with DCM (3 x 20 mL) and
all the organic layers were combined. The DCM layer was then washed with
1 x 50 mL of saturated sodium bicarbonate, water ( 2 x 50 mL) and dried
(sodium sulfate). Removal of the solvent yielded a colorless solid (Compound
C) that was taken to the next step without further purification. Yield: 0.51 g
(91 %). 1H NMR (CDCI3): d 5.3 (s, 2H, Ar-CH ), 6.6 (d, 2H, Ar-H, j =1.0 Hz),
7.4 (m, SH, Ar-H, J= 1.0 Hz) and 7.9 (d, 2H, Ar-H, J= 1.0 Hz).
C. 4-(2-Chloroacetyl)aminobenzyl benzoate Compound D (FIG. 23A~
[00326] The amine (0.51 g, 2.2 mmol) was dissolved in dry dimethylacetamide (
5.0
mL) and cooled in ice. Chloroacetyl chloride (0.28 g, 2.5 mmol) was added
dropwise via a syringe and the solution was allowed to come to RT and stirred
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for 2h. An additional, 2.5 mmol of chloroacetyl chloride was added and
stirring was continued for 2 h more. The reaction mixture was then poured
into ice cold water (100 mL). The precipitated solid was filtered and washed
with water and then recrystallized from hexane/ether to yield a colorless
solid
(Compound D). Yield: 0.38 g (56%). 'H NMR (CDCl3): d 4.25 (s, 2H, CH -
CI), 5.4 (s, 2H, Ar-H), 7.4 (m, 5H, Ar-H), 7.6 (d, 2H, Ar-H) , 8.2 (d, 2H, Ar-
H) and 8.4 (s, 1 H, -CONH).
[00327] text-Butyl2-{1,4,7,10-tetraaza-7,10-bis~[(tert-
butyl)oxycarbonyl]methyl}-4-
[(N-~4-[benzyloxycarbonyl]phenyl } carbamoyl] cyclododecyl } acetate,
Compound E (FIG. 23A):
[00328] DO3A-tri-t-butyl ester.HCl (5.24 g, 9.5 mmol) was suspended in 30.0 mL
of
dry acetonitrile and anhydrous potassium carbonate (2.76 g, 20 mmol) was
added and stirred for 30 min. The chloroacetamide D (2.8 g, 9.2 mmol) in dry
acetonitrile (20.0 mL) was then added dropwise to the above mixture for 10
min. The reaction mixture was then stirred overnight. The solution was
filtered and then concentrated under reduced pressure to a paste. The paste
was dissolved in about 200.0 mL of water and extracted with 5 x 50 mL of
ethyl acetate. The combined organic layer was washed with water (2 x 100
mL) and dried (sodium sulfate). The solution was filtered and evaporated
under reduced pressure to a paste and the paste was chromatographed over
flash silica gel (600.0 g). Elution with 5% methanol in DCM eluted the
product. All the fractions that were homogeneous on TLC were pooled and
evaporated to yield a colorless gum. The gum was recrystallized from
isopropylether and DCM to prepare Compound E. Yield: 4.1 g (55%). 1H
NMR (CDCl3): d 1.5 (s, 27H, methyls), 2.0 - 3.75 (m, 24H, NCH s), 5.25 (d,
2H, Ar-CHa), 7.3 (m, 5H, Ar-H), 7.8 (d, 2H, Ar-H) and 7.95(d, 2H, Ar-H). M.
S. - m/z 804.3 [M+H].
D. Reduction of the above acid E to prepare Compound F FIG. 23A):
[00329] The benzyl ester E from above (1.0 g, 1.24 mmol) was dissolved in
methanol-
water mixture (10.0 mL, 95:5) and palladium on carbon was added (10%, 0.2
g). The solution was then hydrogenated using a Parr apparatus at 50.0 psi for
8h. The solution was filtered off the catalyst and then concentrated under
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reduced pressure to yield a colorless fluffy solid F. It was not purified
further
and was taken to the next step immediately. MS: m/z 714.3 [M+NaJ.
E. Preparation of L203 (FIG. 23B)
[00330) The above acid F was coupled to the amine on the resin [H-Q(Trt)-
W(Boc)-A-
V-G-H(Trt)-L-M-Resin) Resin A and F from above using standard coupling
procedures described above. 0.5 g (0.2 mmol) of the resin yielded 31.5 mg of
the final purified peptide (10.9%) N [(313,513,12a)-3-[[[4,7,10-
Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-ylJ acetyl) amino)-4-
aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-
L-leucyl-L-methioninamide (L203) (FIG. 23B).
Example XVII - Figure 24
Synthesis of L204
[00331) Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-L-M-resin (0.5 g, 0.2 mmol) (Resin A)
was
used. Fmoc-Gly-OH was loaded first followed by F from the above procedure
(FIG. 23A)
employing standard coupling conditions. Yield: 24.5 mg (8.16%) of N
[(313,513,12a)-3-
[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-ylJacetyl]amino)-4-
aminobenzoyl-glycyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-
histidyl-L-
leucyl-L-methioninamide (L204) (FIG. 24).
Example XVIII - Figure 25
Synthesis of L205
[00332) Fmoc-6-aminonicotinic acid' was prepared as described in the
literature
("Synthesis of diacylhydrazine compiounds for therapeutic use." Hoelzemann,
G.; Goodman,
S. (Merck Patent G.m.b.H.., Germany). Ger.Offen. 2000, 16 pp. CODEN: GWXXBX DE
19831710 A1 20000120) and coupled with preloaded Fmoc-Q(Trt)-W(Boc)-A-V-G-
H(Trt)-
L-M-resin (0.5 g, 0.2 mmol) Resin A, followed by the other amino groups as
above to
prepare N [(313,513,12a)-3-[[ [4,7,10-Tris(carboxymethyl)-1,4,7,10-
tetraazacyclododec-1-
ylJ acetyl] amino)-4-aminobenzoyl-glycyl-L-glutaminyl-L-tryptophyl-L-al anyl-L-
valyl-glycyl-
L-histidyl-L-leucyl-L-methioninamide (L205) Yield: 1.28 mg (0.4%).
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Example XIX - Figures 26A and 26B
Synthesis of L206
A. 4'-Fmoc-amino-3'-methylbinhenyl-4-carboxylic acid B'
[00333] The amino acid (0.41 g, 1.8 mmol) was dissolved in a solution of
cesium
carbonate (0.98 g, 3.0 mmol) in 10.0 mL of water. See "Rational Design of
Diflunisal Analogues with Reduced Affinity for Human Serum Albumin "
Mao, H. et al J. Am. Chem. Soc., 2001, 123(43), 10429-10435. This solution
was cooled in an ice bath and a solution of Fmoc-Cl (0.52 g, 2.0 mmol) in
THF (10.0 mL) was added dropwise with vigorous stirring. After the addition,
the reaction mixture was stirred at RT for 20h. The solution was then
acidified with 2N HCI. The precipitated solid was filtered and washed with
water (3 x 20 mL) and air dried. The crude solid was then recrystallized from
acetonitrile to yield a colorless fluffy solid B (FIG. 26A). Yield: 0.66 g
(75%). 'H NMR (DMSO-d6): d 2.2 (s, Ar-Me), 4.25 (t, 1H, Ar-CH, j = SHz),
4.5 (d, 2H, O-CH2, j = 5.0 Hz), 7.1 (bs, 1H, CONH), 7.4 - 8.0 (m, 8H, Ar-H)
and 9.75 (bs, 1H, -COOH). M. S.: m/z 472.0 [M-H].
[00334] The acid B from above was coupled to Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-
L-M-resin (0.2g, 0.08 mmol) resin A with the standard coupling conditions.
Additional groups were added as above to prepareN [(313,513,12a.)-3-
[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-
yl]acetyl]amino]acetyl]amino]- [4'-Amino-2'-methyl biphenyl-4-carboxyl]-L-
glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-
methioninamide (L206). Yield: 30.5 mg (24%).
Example XX - Figures 27A-B
Synthesis of L207
[00335] 3'-Fmoc-amino-biphenyl-3-carboxylic acid was prepared from the
corresponding
amine using the procedure described above. See "Synthesis of 3'-methyl-4-'-
nitrobiphenylcarboxylic acids by the reaction of 3-methyl-4-
nitrobenzenenediazonium acetate
with methyl benzoate", Boyland, E. and Gorrod, J., J. Chem. Soc., Abstracts
(1962), 2209-
11. 0.7G of the amine yielded 0.81 g of the Fmoc-derivative (58%) (Compound B,
FIG.
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27A). 'H NMR (DMSO-db): d 4.3 (t, 1H, Ar-CH), 4.5 (d, 2H, O-CH2), 7.25-8.25
(m, 16H,
Ar-H) and 9.9 (s, 1 H, -COOH). M. S. - m/z 434 [M-H].
[00336] Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-L-M-resin (0.2g, 0.08 mmol) resin A
was
coupled to the above acid B and additional groups as above (FIG. 27B). 29.0 mg
ofN
[(3J3,513,12a)-3-[[[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-
yl]acetyl]amino]acetyl]amino]- [3'-amino-biphenyl-3-carboxyl]-L-glutaminyl-L-
tryptophyl-
L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L207) was
prepared (23%).
Example XXI - Fi ug re 28
Synthesis of L208
[00337] Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-L-M-resin (0.2g, 0.08 mmol) A was
deblocked and coupled to terephthalic acid employing HATU as the coupling
agent. The
resulting acid on the resin was activated with DIC and NHS and then coupled to
ethylenediamine. DOTA-mono acid was finally coupled to the amine on the resin.
N
[(313,513,12x,)-3-[[ [ [[4,7,10-Tris(carboxymethyl)-1,4,7,10-
tetraazacyclododec-1-
yl]acetyl]amino]acetyl]amino]- [1,2-diaminoethyl-terephthalyl]-L-glutaminyl-L-
tryptophyl-
L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide (L208) was
prepared for a
yield of 17.5 mg (14%)
Example XXII - Figures 29A-B
Synthesis of L209
A. Boc-Glu(G-OBn)-G-OBn:
[00338] Boc-Glutamic acid (5.0 g, 20.2 mol) was dissolved in THF (50.0 mL) and
cooled to 0°C in an ice bath. HATU (15.61 g, 41.0 mmol) was added
followed
by DIEA (6.5 g, 50.0 mmol). The reaction mixture was stirred at 0°C for
30
min. Benzyl ester of glycine [8.45 g, 50 mmol, generated from neutralizing
benzyl glycine hydrochloride with sodium carbonate and by extraction with
DCM and solvent removal] was added in THF (25.0 mL). The reaction
mixture was allowed to come to RT and stirred for 20h at RT. All the
volatiles were removed under reduced pressure. The residue was treated with
saturated sodium carbonate solution (100 mL) and extracted with ethyl acetate
(3 x 100 mL). The organic layers were combined and washed with 1N HCl (2
x 100 mL) and water (2 x 100 mL) and dried (sodium sulfate). The solution
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was filtered and solvent was removed under reduced pressure to yield a paste
that was chromatographed over flash silica gel (500.0 g). Elution with 2%
methanol in DCM yielded the product as a colorless paste (Compound B, FIG.
29A). Yield: 8.5 g (74.5%). 1H NMR (CDCl3): d 1.4 (s, 9H, -CH3s), 2.0 - 2.5
(m, 4H,-CH-CHZ and CO-CH), 4.2 (m, SH, N-CH -CO), 5.15 (s, 4H, Ar-
CH ), 5.45 (bs, 1 H, Boc-NH), 7.3 (m, 1 OH, Ar-H) and 7.6 (2bs, 2H, CONH).
M. S. - mlz 564.1 [M+H]. Analytical HPLC retention time - 8.29 min (>97%
pure, 20-65%B over 15 min).
B. H-GIu~G-OBn)-G-OBn:
[00339] The fully protected glutamic acid derivative (1.7 g, 3.2 mmol) B from
above
was dissolved in DCM/TFA (4:1, 20 mL) and stirred until the starting material
disappeared on TLC (2 h). The reaction mixture was poured into ice cold
saturated sodium bicarbonate solution (200 mL) and the organic layer was
separated and the aqueous layer was extracted with 2 x 50 mL of DCM and
combined with the organic layer. The DCM layer was washed with saturated
sodium bicarbonate (2 x 100 mL), water (2 x 100 mL) and dried (sodium
sulfate). The solution was filtered and evaporated under reduced pressure and
the residue was dried under vacuum to yield a glass (Compound C, FIG. 29A)
that was taken to the next step without further purification. Yield: 0.72 g
(95%). M. S. - m/z 442.2 [M+H].
C. ~DOTA-tri-t-butyl -GIu-(G-OBn)-G-OBn:
[00340] The amine C from above (1.33 g, 3 mmol) in anhydrous DCM (10.0 mL) was
added to an activated solution of DOTA-tri-t-butyl ester [2.27 g, 3.6 mmol
was treated with HBTU, 1.36 g, 3.6 mmol and DIEA 1.04 g, 8 mmol and
stirred for 30 min at RT in 25 mL of dry DCM] and stirred at RT for 20h].
The reaction mixture was diluted with 200 mL of DCM and washed with
saturated sodium carbonate (2 x 150 mL) and dried (sodium sulfate). The
solution was filtered and solvent was removed under reduced pressure to yield
a brown paste. The crude product was chromatographed over flash silica gel
(500.0 g). Elution with 2% methanol in DCM furnished the product as a
colorless gum (Compound D, FIG. 29A). Yield: 1.7 g (56.8%). IH NMR
(CDCl3): d 1.3 and 1.4 (2s, 9H, three methyls each from the free base and the
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sodium adduct of DOTA), 2.0 - 3.5 (m, 20H, N-CH2s and -CH-CH and CO-
CH2), 3.75 - 4.5 (m, 13H, N-CH -CO), 5.2 (m, 4H, Ar-CHZ) and 7.25 (m,
1 OH, Ar-H). M. S. m/z -1 Ol 8.3 [M+Na] and 996.5 [M+H] and 546.3
[M+Na+H]/2. HPLC - Retention Time: 11.24 min (>90% , 20-80% B over 30
min).
D. ~DOTA-tri-t-butt -GIu~G-OH)-G-OH:
[00341 ] The bis benzyl ester (0.2 g, 0.2 mmol) D from above was dissolved in
methanol-water (20 mL, 9:1 ) and hydrogenated at 50 psi in the presence of
10% Pd/C catalyst (0.4 g, 50% by wt. water). After the starting material
disappeared on HPLC and TLC (4h), the solution was filtered off the catalyst
and the solvent was removed under reduced pressure and the residue was dried
under high vacuum for about 20h (<0.1 mm) to yield the product as a colorless
foam (Compound E, FIG. 29A). Yield: 0.12 g (73.5%). 'H NMR (DMSO-
d6): d 1.3 and 1.4 (2s, 9H corresponding to methyls of free base and the
sodium adduct of DOTA), 1.8 - 4.7 (m, 33H, NCH s, COCH and CH-CH2
and NH-CH-CO), 8.1, 8.2 and 8.4 (3bs, NHCO). M. S.: m/z - 816.3 [M+H]
and 838.3 [M+Na]. HPLC Retention Time: 3.52 min (20-80% B over 30 min,
> 95% pure).
E. H-8-amino-3,6-dioxaoctanoyl-8-amino-3,6-dioxaoctanoyl-Gln-Trp-Ala-Val-
Gly-His-Leu-Met NHS:
[00342] Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-L-M-resin (0.5 g, 0.2 mmol) A was
deblocked and coupled twice sequentially to 8-amino-3,6-dioxaoctanoic acid
to yield the above deprotected peptide (Compound F, FIG. 29B) after
preparative HPLC purification. Yield: 91.0 mg (37%).
[00343] HPLC Retention Time: 8.98 min (>95% purity, 10-40% B in over 10 min).
M. S.: m/z - 1230.6 [M+H], 615.9 [M+2H]/2.
F. Solution phase coupling of the bis-acid E and the amine F from above: (FIG.
29B
[00344] The bis-acid (13.5 mg, 0.0166 mmol) E was dissolved in 100~L of dry
acetonitrile and treated with NHS (4.0 mg, 0.035 mmol) and DIC (5.05 mg,
0.04 mmol) and stirred for 24h at RT. To the above activated acid, the free
amine F (51.0 mg, 0.41 mmol)[generated from the TFA salt by treatment with
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saturated sodium bicarbonate and freeze drying the solution to yield the amine
as a fluffy solid] was added followed by 1 OO~L of NMP and the stirring was
continued for 40h more at RT. The solution was diluted with anhydrous ether
(10 mL) and the precipitate was collected by centrifugation and washed with 2
x 10 mL of anhydrous ether again. The crude solid was then purified by
preparative HPLC to yield the product as a colorless fluffy solid L209 as in
FIG. 29B with a yield of 7.5 mg (14.7%).
Example XXIII - Figures 30A-B
Synthesis of L210
A. H-8-aminooctanoyl-8-aminooctanoyl-Gln-Tm-Ala-Val-Gly-His-Leu-Met-
NHS:
[00345] This was also prepared exactly the same way as in the case of Compound
F
(FIG. 29B), but using 1-aminooctanoic acid and the amine (Compound B,
FIG. 30A) was purified by preparative HPLC. Yield: 95.0 mg (38.9%).
HPLC Retention Time: 7.49 min (>95% purity; 10-40%B over 10.0 min). M.
S.: m/z -1222.7 [M+H], 611.8 [M+2H]/2.
[00346] (DOTA-tri-t-butyl)-Glu-(G-OH)-G-OH (0.0163 g, 0.02 mmol) was converted
to its bis-NHS ester as in the case of L209 in 100~L of acetonitrile and
treated
with the free base, Compound B (60.0 mg, 0.05 mmol) in 1 OO~L of NMP and
the reaction was continued for 40h and then worked up and purified as above
to prepare L210 (FIG. 30B) for a yield of 11.0 mg (18%).
Examine XXIV - Fi ug re 31
Synthesis of L211
[00347] Prepared from 0.2 g Of the Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-L-M-resin
(0.08
mmol) using standard protocols. N [(313,513,12a,)-3-[[[4,7,10-
Tris(carboxymethyl)-1,4,7,10-
tetraazacyclododec-1-yl] acetyl] amino]-glycyl-glycyl-4-aminob enzoyl-L-
glutaminyl-L-
tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide L211
was
prepared in a yield of 4.7 mg (3.7%) (FIG. 31).
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Example XXV - Figure 32
Synthesis of L212
[00348] Prepared from Rink Amide Novagel resin (0.47 mmol/g, 0.2 g, 0.094
mmol) by
building the sequence on the resin by standard protocols. N [(313,5]3,12a)-3-
[[[4,7,10-
Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl]amino]-glycyl-4-
aminobenzoyl-L-glutamyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-
leucyl-L-
methioninamide L212 was prepared for a yield of 25.0 mg (17.7%) (FIG. 32).
Example XXVI - Fi ure 33
Synthesis of L213
[00349] Prepared from Fmoc-Met-2-chlorotrityl chloride resin (NovaBioChem,
0.78
mmol/g, 0.26 g, 0.2 mmol) and the rest of the sequence were built using
standard
methodology. N [(313,513,12a)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-
tetraazacyclododec-
1-yl] acetyl] amino]-glycyl-4-aminobenzoyl-L-glutaminyl-L-tryptophyl-L-al anyl-
L-valyl-
glycyl-L-histidyl-L-leucyl-L-methionine L213 was prepared for a yield of 49.05
mg (16.4%)
(FIG.33).
Example XXVII - Figure 34
Synthesis of L214
[00350] Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-L-M-resin (0.2 g, 0.08 mmol) A was
used to
prepare N [(313,513,12a)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-
tetraazacyclododec-1-
yl]acetyl]amino]-glycyl-4-aminobenzoyl-D-phenylalanyl-L-glutaminyl-L-
tryptophyl-L-
alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-methioninamide L214 using standard
conditions.
8.5 mg of the product (6.4%) was obtained (FIG. 34).
Example XXVIII - Fi, ure 35
Synthesis of L215
[00351] Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-L-M-resin (0.2 g, 0.08 mmol) A was
used to
prepare N [(313,S13,12a)-3-[[[4,7,10-Tris(carboxyrnethyl)-1,4,7,10-
tetraazacyclododec-1
yl] acetyl] amino]-glycyl-4-aminob enzoyl-L-glutaminyl-L-arginyl-L-1 eucyl-
glycyl-L-
asparginyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-
leucyl-L-
methioninarnide L215. 9.2 mg (5.5%) was obtained (FIG. 35).
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Example ~I~ - Figure 36
Synthesis of L216
[00352] Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-L-M-resin (0.2 g, 0.08 mmol) A was
used to
prepareN [(313,S13,12a)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-
tetraazacyclododec-I-
yl)acetyl)amino)-glycyl-4-aminobenzoyl-L-glutaminyl-arginyl-L-tyrosinyl-glycyl-
L-
asparginyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-
leucyl-L-
methioninamide L216. 25.0 mg (14.7%) was obtained (FIG. 36).
Example XXX - Figure 37
Synthesis of L217
[00353) Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-L-M-resin A (0.2g, 0.08 mmol) was used
to
prepareN [(313,513,12a,)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-
tetraazacyclododec-1-
yl) acetyl) amino)-glycyl-4-aminobenzoyl-L-glutaminyl-L-lysyl-L-tyrosinyl-
glycyl-L-
glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-
methioninamide
L217. 58.0 mg (34.7%) was obtained (FIG. 37).
Example ~!:~I - Figure 38
Synthesis of L218
[00354) Fmoc-Q(Trt)-W(Boc)-A-V-G-H(Trt)-L-M-resin A (0.2g, 0.08 mmol) was
used.
Fmoc-Lys(ivDde) was employed for the introduction of lysine. After the linear
sequence was
completed, the protecting group of the lysine was removed using 10% hydrazine
in DMF (2 x
10 mL; 10 min each and then washed). The rest of the amino acids were then
introduced
using procedures described in the "general" section to complete the required
peptide
sequence. L218 in FIG. 38 as obtained in a yield of 40.0 mg (23.2%).
Examule ~S;XYII- Figure 39
Synthesis of L219
[00355) 4-Sulfamylbutyryl AM Novagel resin was used (1.1 mrnol/g; 0.5 g; 0.55
mmol).
The first amino acid was loaded on to this resin at -20° C for 20h. The
rest of the sequence
was completed utilizing normal coupling procedures. After washing, the resin
was alkylated
with 20.0 eq. of iodoacetonitrile and 10.0 equivalents of DIEA for 20h. The
resin Was then
drained of the liquids and washed and then cleaved with 2.0 eq. of pentylamine
in 5.0 mL of
THF for 20h. The resin was then washed with 2 x 5.0 mL of THF and all the
filtrates were
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combined. THF was then evaporated under reduced pressure and the residue was
then
deblocked with 10.0 mL of Reagent B and the peptide N [(313,SJ3,12a)-3-
[[[4,7,10-
Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl] acetyl] amino]-glycyl-4-
aminobenzoyl-D-phenylalanyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-glycyl-
L-
S histidyl-L-leucyl-aminopentyl, L219 was purified as previously described.
28.0 mg (2.8%)
was obtained (FIG. 39).
Example XXXIII- Figure 40
Synthesis of L220
[00356] NovaSyn TGR (0.25 mmol/g; 0.15 g, 0.05 mmol) resin A was used to
prepare N
[(313,513,12a,)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-I-
y1] acetyl] amino]-glycyl-4-aminobenzoyl-L-glutaminyl-L-tryptophyl-L-al anyl-L-
valyl-D-
alanyl-L-histidyl-L-leucyl-L-methioninamide, L220. 31.5 mg (41.4%) was
obtained (FIG.
40).
Example ~:XXIV - Figure 41
Synthesis of L221
[00357] NovaSyn TGR (0.25 mmol/g; 0.1 S g, 0.05 mmol) resin A was used to
prepare N
[(3 ~3,5[i,12a)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-
yl]acetyl]amino]-glycyl-4-aminobenzoyl-D-phenylalanyl-L-glutaminyl-L-
tryptophyl-L-
alanyl-L-valyl-glycyl-L-histidyl-L-leucyl-L-leucinamide, L221. 28.0 mg (34.3%)
was
obtained (FIG. 41).
Example XXXV - Figure 42
Synthesis of L222
[00358] NovaSyn TGR (0.25 mmol/g; 0.15 g, 0.05 mmol) resin A was used to
prepare N
[(3 (3,5 (3,12a,)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-
1-
yl]acetyl]amino]-glycyl-4-aminobenzoyl-D-tyrosinyl-L-glutaminyl-L-tryptophyl-L-
alanyl-L-
valyl-betaalanyl-L-histidyl-L-phenylalanyl-L-norleucinamide, L222. 34.0 mg
(40.0%) was
obtained (FIG. 42).
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Example XXXVI - Fi ug~ re 43
Synthesis of L223
[00359] NovaSyn TGR (0.25 mmol/g; 0.15 g, 0.05 mmol) resin A was used to
prepare N
[(3[i,5 J3,12a,)-3-[ [ [4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-
1-
ylJacetyl]amino]-glycyl-4-aminobenzoyl-L-phenylalanyl-L-glutaminyl-L-
tryptophyl-L-
alanyl-L-valyl-betaalanyl-L-histidyl-L-phenylalanyl-L-norleucinamide, L223.
31.2 mg
(37.1 %) was obtained (FIG. 43).
Example ~:XXVII - Figure 44
Synthesis of L224
[00360] NovaSyn TGR (0.25 mmol/g; 0.15 g, 0.05 mmol) resin A was used to
prepare N
[(3[i,5 j3,12oc)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-
yl]acetyl]amino]-glycyl-4-aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-
glycyl-L-
histidyl-L-phenylalanyl-L-leucinamide, L224. 30.0 mg (42.2%) was obtained
(FIG. 44).
Example ~:XXVIII - Figure 45
Synthesis of L225
[00361] NovaSyn TGR (0.25 mmol/g; 0.15 g, 0.05 mmol) resin A was used to
prepare N
[(3 (3,5(3,12a)-3-[[ [4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-
ylJ acetyl] aminoJ-glycyl-4-aminobenzoyl-L-leucyl-L-tryptophyl-L-al anyl-L-
valinyl-glycyl-L-
serinyl-L-phenylalanyl-L-methioninamide, L225. 15.0 mg (20.4%) was obtained
(FIG. 45).
Examule XXXIX - Fi, ug re 46
Synthesis of L226
[00362] NovaSyn TGR (0.25 mmol/g; 0.15 g, 0.05 mmol) resin A was used to
prepare N
[(3 /3,5(3,12a)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-
yl]acetyl]aminoJ-glycyl-4-aminobenzoyl-L-histidyl-L-tryptophyl-L-alanyl-L-
valyl-glycyl-L-
histidyl-L-leucyl-L-methioninamide, L226. 40.0 mg (52.9%) was obtained (FIG.
46).
Example XL - Figure 47
Synthesis of L227
[00363] NovaSyn TGR (0.25 mmol/g; 0.15 g, 0.05 mmol) resin A was used to
prepare N
[(3 [3,5(3,12a.)-3-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-
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yl]acetyl]amino]-glycyl-4-aminobenzoyl-L-leucyl-L-tryptophyl-L-alanyl-L-
threonyll-glycyl-
L-histidyl-L-phenylalanyl-L-methioninamide L227. 28.0 mg (36.7%) was obtained
(FIG.
47).
Example XLI - Figure 48
Synthesis of L228
[00364] NovaSyn TGR (0.25 mmol/g; 0.15 g, 0.05 mmol) resin A was used to
prepare N
[(3 (395 (3,12oc)-3-[ [ [4,7,10-Tris(carboxymethyl)-1,4,7,10-
tetraazacyclododec-1-
yl]acetyl]amino]-glycyl-4-aminobenzoyl-L-glutaminyl-L-tryptophyl-L-alanyl-L-
valyl-glycyl-
L-histidyl-L-phenylalanyl-L-methioninamide, L228. 26.0 mg (33.8%) was obtained
(FIG.
48).
EXAMPLE XLII - Synthesis of Additional GRP Compounds
A. General procedure for the preparation of 4,4'-
Aminomethylbiphenylcarboxylic acid (B2) and 3,3'-
aminometh~phenylcarboxylic acid (B3):
1. Meths d~ymethylbiphenylcarbox 1
Commercially available (Aldrich Chemical Co.) 4-
hydroxymethylphenylboric acid or 3-hydroxymethylphenylboric acid
(1.0 g, 6.58 mmol) was stirred with isopropanol (10 mL) and 2M
sodium carbonate (16 mL) until the solution became homogeneous.
The solution was degassed by passing nitrogen through the solution
and then treated with solid methyl-3-bromobenzoate, or methyl-4-
bromobenzoate (1.35 g, 6.3 mmol) followed by the Pd (0) catalyst
f [(C6I-is)3P]4Pd; 0.0238, 0.003 mmol~. The reaction mixture was kept
at reflux under nitrogen until the starting bromobenzoate was
consumed as determined by TLC analysis (2-3 h). The reaction
mixture was then diluted with 250 mL of water and extracted with
ethyl acetate (3 x 50 mL). The organic layers were combined and
washed with saturated sodium bicarbonate solution (2 x 50 mL) and
dried (Na2SO4). The solvent was removed under reduced pressure and
the residue was chromatographed over flash silica gel (100 g). Elution
with 40% ethyl acetate in hexanes yielded the product either as a solid
or oil.
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Yield:
B2 -0.45g (31 %); m. p. - 170-171 ° C.
B3 - 0.69 g (62%); oil.
'H NMR (CDCl3) d B2- 3.94 (s,3H, -COOCH3), 4.73 (s, 2H, -CHZ-Ph), 7.475 (d,
2H, J=
SHz), 7.6 (d, 2H, J = 10 Hz), 7.65 (d, 2H, J = SHz) and 8.09 (d, 2H, J = 10
Hz).
Ii~I. S. - m/e - 243.0 [M+H]
B3 - 3.94 (s, 3H, -COOCH3), 4.76 (s, 2H, -CHZ-Ph), 7.50 (m, 4H), 7.62 (s, 1
H), 7.77 (s, 1 H),
8.00 (s, 1 H) and 8.27 (s, 1 H).
M. S. - m/e - 243.2 [M+H]
2. Azidomethylbiphenyl carboxylates:
[00365] The above biphenyl alcohols (2.0 mmol) in dry dichloromethane (10 mL)
were
cooled in ice and treated with diphenylphosphoryl azide (2.2 mol) and DBU
(2.0 mmol) and stirred under nitrogen for 24h. The reaction mixture was
diluted with water and extracted with ethyl acetate (2 x 25 mL). The organic
layers were combined and washed successfully with 0.5 M citric acid solution
(2 x 25 mL), water (2 x 25 mL) and dried (Na2S04). The solution was filtered
and evaporated under reduced pressure to yield the crude product. The 4,4'-
isomer was crystallized from hexane/ether and the 3,3'-isomer was triturated
with isopropyl ether to remove all the impurities; the product was
homogeneous as determined on TLC analysis and further purification was not
required.
Yield:
Methyl-4-azidomethyl-4-biphenylcaroxylate- 0.245 g (46%); m. p. -106-
108° C.
Methyl-4-azidomethyl-4-biphenylcaroxylate - 0.36 g (59%, oil)
1H NMR (CDCl3) d - 4,4'-isomer - 3.95 (s, 3H, -COOCH3), 4.41 (s, 2H, -CHZN3),
7.42 (d,
2H, J = 5 Hz), 7.66 (m, 4H) and 8.11 (d, 2H, J = 5 Hz)
3,3'-Isomer - 3.94 (s, 3H, -COOCH3), 4.41 (s, 2H, -CH2N3), 7.26-7.6 (m, SH),
7.76 (d, 1 H, J
=10 Hz), 8.02 (d, 1 H, J = 5 Hz) and 8.27 (s, 1 H).
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3. Hydrolysis of the methyl esters of biphenylcarbox lates:
[00366] About 4 mmol of the methyl esters were treated with 20 mL of 2M
lithium
hydroxide solution and stirred until the solution was homogeneous (20-24 h).
The aqueous layer was extracted with 2 x 50 mL of ether and the organic layer
was discarded. The aqueous layer was then acidified with 0.5 M citric acid
and the precipitated solid was filtered and dried. No other purification was
necessary and the acids were taken to the next step.
Yield:
4,4'-isomer - 0.87 g of methyl ester yielded 0.754 g of the acid (86.6%); m.
p. - 205-210° C
3,3'-isomer - 0.48 g of the methyl ester furnished 0.34 g of the acid (63.6%);
m. p. -102-
105° C.
'H NMR (DMSO-d6) d : 4,4'-isomer- 4.52 (s, 2H, -CH2N3), 7.50 (d, 2H, J = 5
Hz), 7.9 (m,
4H), and 8.03 (d, 2H, J = 10 Hz)
3,3'-isomer- 4.54 (s, 2H, -CHaN3), 7.4 ( d, 1H, J = 10 Hz), 7.5-7.7 (m, 4H),
7.92 (ABq, 2H)
and 8.19 (s, 1 H).
4. Reduction of the azides to the amine:
[00367] This was carried out on the solid phase and the amine was never
isolated. The
azidocarboxylic acid was loaded on the resin using the standard peptide
coupling protocols. After washing, the resin containing the azide was shaken
with 20 equivalents of triphenylphosphine in THF/water (95:5) for 24 h. The
solution was drained under a positive pressure of nitrogen and then washed
with the standard washing procedure. The resulting amine was employed in
the next coupling.
5. (3(3, 5(3, 7a., l2oc)-3-[ f (9H-Flouren-
9ylmethoxy)amino] acetyl ~ amino-7,12-dih,~ycholan-24-oic
[00368] Tributylamine (3.2 mL); 13.5 mmol) was added dropwise to a solution of
Fmoc-glycine (4.0 g, 13.5 mmol) in THF (80 mL) stirred at 0° C.
Isobutylchloroformate (1.7 mL; 13.5 mmol) was subsequently added and, after
10 min, a suspension of tributylamine (2.6 mL; 11.2 mmol) and (3(3, 5(3, 7a,
12a)-3-amino-7,12-dihydroxycholan-24-oic acid (4.5 g; 11.2 mmol) in DMF
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(80 mL) was added dropwise, over 1 h, into the cooled solution. The mixture
was allowed to warm up to ambient temperature and after 6 h, the solution was
concentrated to 120 mL, then water (180 mL) and 1N HCI (30 mL) were
added (final pH 1.5). The precipitated solid was filtered, washed with water
(2
x 100 mL), vacuum dried and purified by flash chromatography. Elution with
chloroform/methanol (8:2) yielded the product as a colorless solid.
Yield: 1.9 g (25%). TLC: Rf 0.30 (CHCl3/MeOH/NH40H - 6:3:1).
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IN VITRO AND IN VIVO TESTING OF COMPOUNDS
Example XLIII: IsZ vitro Binding Assay for GRP Receptors in PC3 Cell Lines
FIGS. 14 A-B
[00369] To identify potential lead compounds, an in vitro assay that
identifies compounds
with high affinity for GRP-R was used. Since the PC3 cell Line, derived from
human prostate
cancer, is known to exhibit high expression of GRP-R on the cell surface, a
radio ligand
binding assay in a 96-well plate format was developed and validated to measure
the binding
of I25I-BBN to GRP-R positive PC3 cells and the ability of the compounds of
the invention to
inhibit this binding. This assay was used to measure the ICSO for RP527
ligand, DO3A-
monoamide-Aoc-QWAVGHLM-NH2 (controls) and compounds of the invention which
inhibit the binding of l2sl-BBN to GRP-R. (RP527 =N,N-dimethylglycine-Ser-
Cys(Acm)-
Gly-5-aminopentanoic acid-BBN (7-14) (SEQ. ID.NO: 1], which has MS = 1442.6
and IC50
0.84). Van de Wiele C, Dumont F et al., Technetium-99m RP527, a GRP analogue
for
visualization of GRP receptor-expressing malignancies: a feasibility study.
Eur.J.Nucl.Med.
27; 1694-1699 (2000). D03A-rnonoamide-Aoc-QWAVGHLM-NHa is also referred to as
D03A-monoamide-8-amino-octanoic acid-BBN (7-14) (SEQ. ID. NO: 1], and has
MS=1467Ø DO3A monoamide-aminooctanyl-BBN[7-14].
[00370] The Radioligand Binding Plate Assay was validated for BBN and BBN
analogues
(including commercially available BBN and Ll) and also using 99mTc RP527 as
the
radioligand.
A. Materials and Methods:
1. Cell culture:
[00371 ] PC3 (human prostate cancer cell line) were obtained from the American
Type
Culture Collection and cultured in RPMI 1640 (ATCC) in tissue culture flasks
(Corning). This growth medium was supplemented with 10% heat inactivated
FBS (Hyclone, SH30070.03), 10 mM HEPES (GibcoBRL, 15630-080), and
antibiotic/antimycotic (GibcoBRL, 15240-062) for a final concentration of
penicillin-streptomycin (100 units/mL), and fungizone (0.25 ~g/mL). All
cultures were maintained in a humidified atmosphere containing 5% COa/95%
air at 37°C, and passaged routinely using 0.05% trypsin/EDTA (GibcoBRL
25300-054) where indicated. Cells for experiments were plated at a
concentration of 2.0x104 /well either in 96-well white /clear bottom
microtiter
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plates (Falcon Optilux-I) or 96 well black/clear collagen I cellware plates
(Beckton Dickinson Biocoat). Plates were used for binding studies on day 1
or 2 post-plating.
2. Binding buffer:
[00372] RPMI 1640 (ATCC) supplemented with 20mM HEPES, 0.1 % BSA (w/v), 0.5
mM PMSF (AEBSF), bacitracin (50 mg/S00 ml), pH 7.4. ~25I-BBN (carrier
free, 2200 Ci/mmole) was obtained from Perkin-Elmer.
B. Competition assay with ~ZSI-BBN for GRP-R in PC3 cells:
[00373] A 96-well plate assay was used to determine the ICSn of various
compounds of
the invention to inhibit binding of ~25I-BBN to human GRP-R. The following
general procedure was followed:
[00374] All compounds tested were dissolved in binding buffer and appropriate
dilutions were also done in binding buffer. PC3 cells (human prostate cancer
cell line) for assay were plated at a concentration of 2.0x 104 /well either
in 96-
1 S well white /clear bottomed microtiter plates (Falcon Optilux-I) or 96 well
black/clear collagen I cellware plates (Beckton Dickinson Biocoat). Plates
were used for binding studies on day 1 or 2 post-plating. The plates were
checked for confluency (>90% confluent) prior to assay. For the assay, RP527
or D03A-monoamide-Aoc-QWAVGHLM-NH2 ligand, (controls), or
compounds of the invention at concentrations ranging from 1.25 x 10-9 M to
SxlO-9M, was co-incubated with jZSI-BBN (25,000 cpm/well). These studies
were conducted with an assay volume of 75 ~1 per well. Triplicate wells were
used for each data point. After the addition of the appropriate solutions,
plates
were incubated for 1 h at 4°C to prevent internalization of the ligand-
receptor
complex. Incubation was ended by the addition of 200 ~,l of ice-cold
incubation buffer. Plates were washed 5 times and blotted dry. Radioactivity
was detected using either the LKB CompuGamma counter or a microplate
scintillation counter.
[00375] Competition binding curves for RP527 (control) and L70, a compound of
the
invention can be found in FIGS. 14A-B. These data show that the IC50 of the
RP527 control
is 2.SnM and that of L70, a compound of this invention is SnM. The IC50 of the
D03A-
monoamide-Aoc-QWAVGHLM-NH2 control was SnM. IC50 values for those compounds of
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the invention tested can be found in Tables 1-3, supra, and show that they are
comparable to
that of the controls and thus would be expected to have sufficient affinity
for the receptor to
allow uptake by receptor bearing cells in vivo.
C. Internalization & Efflux assay:
[00376] These studies were conducted in a 96-well plate. After washing to
remove
serum proteins, PC3 cells were incubated with lzsl-BBN, ~~~Lu-D03A-
monoamide-Aoc-QWAVGHLM-NH2 or radiolabeled compounds of this
invention for 40 min, at 37 °C. Incubations were stopped by the
addition of
200 ~.l of ice-cold binding buffer. Plates were washed twice with binding
buffer. To remove surface-bound radioligand, the cells were incubated with
0.2M acetic acid (in saline), pH 2.~ for 2 min. Plates were centrifuged and
the
acid wash media were collected to determine the amount of radioactivity
which was not internalized. The cells were collected to determine the amount
of internalized l2sI-BBN, and all samples were analyzed in the gamma
counter. Data for the internalization assay was normalized by comparing
counts obtained at the various time points with the counts obtained at the
final
time point (T40 min).
[00377) For the efflux studies, after loading the PC3 cells with l2sl-BBN or
radiolabeled compounds of the invention for 40 min at 37°C, the unbound
material was filtered, and the % of internalization was determined as above.
The cells were then resuspended in binding buffer at 37°C for up to
3h. At
0.5, 1, 2, or 3 h, the amount remaining internalized relative to the initial
loading level was determined as above and used to calculate the percent efflux
recorded in Table 5.
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TABLE 5
Internalisation and efflux of ~25I-BBN and the Lu-177 complexes of D03A-
monoamide-Aoc-
QWAVGHLM-NHZ (control) and compounds of this invention
I-BBN D03A-monoamide- L63 L64 L70
Aoc-QWAVGHLM-
NHZ (control
Internalisation (40 59 89 64 69 70
minutes)
Efflux (2h) 35 28 0 20 12
These data show that the compounds of this invention are internalized and
retained by the
PC3 cells to a similar extent to the controls.
Example XLIV - Preparation of Tc-labeled GRP compounds.
[00378] Peptide solutions of compounds of the invention identified in Table 6
were
prepared at a concentration of 1 mg/mL in 0.1 % aqueous TFA. A stannous
chloride solution
was prepared by dissolving SnCl2~2H2O (20 mg/mL) in 1 N HCl. Stannous
gluconate
solutions containing 20 ~.g of SnCh~2HaO/100 ~L were prepared by adding an
aliquot of the
SnCl2 solution (10 ~L) to a sodium gluconate solution prepared by dissolving
13 mg of
sodium gluconate in water. A hydroxypropyl gamma cyclodextrin [HP-y-CD]
solution was
prepared by dissolving 50 mg of HP-y-CD in 1 mL of water.
[00379] The 99mTc labeled compounds identified below were prepared by mixing
20 ~L of
solution of the unlabeled compounds (20 fig), 50 ~L of HP-y-CD solution, 100
~.L of Sn-
gluconate solution and 20 to 50 ~.L Of 9smTc pertechnetate (5 to 8 mCi,
Syncor). The final
volume was around 200 ~L and final pH was 4.5 - 5. The reaction mixture was
heated at
100°C for 15 to 20 min. and then analyzed by reversed phase HPLC to
determine
radiochemical purity (RCP). The desired product peaks were isolated by HPLC,
collected
into a stabilizing buffer containing 5 mg/mL ascorbic acid, 16 mg/mL HP-y-CD
and 50 mM
phosphate buffer, pH 4.5, and concentrated using a speed vacuum to remove
acetonitrile.
The HPLC system used for analysis and purification was as follows: C18 Vydac
column, 4.6
x 250 mm, aqueous phase: 0.1 % TFA in water, organic phase: 0.085% TFA in
acetonitrile.
Flow rate: 1 mL/min. Isocratic elution at 20% - 25% acetonitrile/0.085% TFA
was used,
depending on the nature of individual peptide.
[00380] Labeling results are summarized in Table 6.
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TABLE 6
HPLC
retentionInitial RCP4 (%)
time RCP3 immediately following
om oundl e uence2 min % urification
L2 -RJQWAVGHLM-NHZ 5.47 89.9 95.6
L4 -SJQWAVGHLM- NHZ 5.92 65 97
L8 -JKQWAVGHLM- NH2 6.72 86 94
Ll -KJQWAVGHLM- NH2 5.43 88.2 92.6
L9 -JRQWAVGHLM- NH2 7.28 91.7 96.2
L7 -aJQWAVGHLM- NH2 8.47 88.6 95.9
n.d. = not detected
1: All compounds were conjugated with an N,N'-dimethylglycyl-Ser-Cys-Gly metal
chelator.
The Acm protected form of the ligand was used. Hence, the ligand used to
prepare the
99mTc complex of L2 was N,N'-dimethylglycyl-Ser-Cys (Acm)-Gly-RJQWAVGHLM-NHa.
The Acm group was removed during chelation to Tc.
2: In the Sequence, "J" refers to 8-amino-3,6-dioxaoctanoic acid and "a"
refers to D-alanine.
3: Initial RCP measurement taken immediately after heating and prior to HPL
purification.
4: RCP determined following HPLC isolation and acetonitrile removal via speed
vacuum.
Example XLV - Preparation of i~~Lu-L64 for cell binding and biodistribution
studies:
[00381] This compound was synthesized by incubating IO ~.g L64 ligand (10 ~,L
of a 1
mg/mL solution in water), 100 ~L ammonium acetate buffer (0.2M, pH 5.2) and ~1-
2 mCi of
~~~LuCl3 in O.OSN HCl (MURK) at 90°C for 15 min. Free ~~~Lu was
scavenged by adding 20
~,L of a I % Na2EDTA~2H20 (Aldrich) solution in water. The resulting
radiochemical purity
(RCP) was N95%. The radiolabeled product was separated from unlabeled ligand
and other
impurities by HPLC, using a YMC Basic C8 column [4.6 x 150 mm], a column
temperature
of 30°C and a flow rate of 1 mL/min, with a gradient of 68%A/32%B to
66%A/34%B over
30 min., where A is citrate buffer (0.02M, pH 3.0), and B is 80% CH3CN/20%
CH30H. The
isolated compound had an RCP of 100% and an HPLC retention time of 23.4
minutes.
[00382] Samples for biodistribution and cell binding studies were prepared by
collecting
the desired HPLC peak into 1000 ~L of citrate buffer (0.05 M, pH 5.3,
containing 1
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ascorbic acid, and 0.1 % HSA). The organic eluent in the collected eluate was
removed by
centrifugal concentration for 30 min. For cell binding studies, the purified
sample was
diluted with cell-binding media to a concentration of 1.5 ~Ci/mL within 30
minutes of the in
vitro study. For biodistribution studies, the sample was diluted with citrate
buffer (0.05 M,
pH 5.3, containing 1 % sodium ascorbic acid and 0.1 % HSA) to a fnal
concentration of 50
~Ci/mL within 30 minutes of the in vivo study.
Example XLVI - Preuaration of I~~Lu-L64 for radiotherapy studies:
[00383] This compound was synthesized by incubating 70 qg L64 ligand (70 ~L of
a 1
mg/mL solution in water), 200 ~.L ammonium acetate buffer (0.2M, pH 5.2) and
~30 - 40
mCi of l~~LuCl3 in O.OSN HCl (MURR) at 85°C for 10 min. After cooling
to room
temperature, free ~~~Lu was scavenged by adding 20 ~L of a 2% Na2EDTA~2H20
(Aldrich)
solution in water. The resulting radiochemical purify (RCP) was ~95%. The
radiolabeled
product was separated from unlabeled ligand and other impurities by HPLC,
using a 300VHP
Anion Exchange column (7.5 x 50 mm) (Vydac) that was sequentially eluted at a
flow rate of
1 mL/min with water, 50% acetonitrile/water and then 1 g/L aqueous ammonium
acetate
solution. The desired compound was eluted from the column with 50% CH3CN and
mixed
with ~l mL of citrate buffer (0.05 M, pH 5.3) containing 5% ascorbic acid,
0.2% HSA, and
0.9% (v:v) benzyl alcohol. The organic part of the isolated fraction was
removed by spin
vacuum for 40 min, and the concentrated solution (~20-25 mCi) was adjusted
within 30
minutes of the in vivo study to a concentration of 7.5 mCi/mL using citrate
buffer (0.05 M,
pH 5.3) containing 5% ascorbic acid, 0.2% HSA, and 0.9% (v:v) benzyl alcohol.
The
resulting compound had an RCP of >95%.
Examule XLVII - Preparation of lilln-L64:
[00384] This compound was synthesized by incubating 10 ~g L64 ligand (5 ~L of
a 2
mg/mL solution in 0.01 N HCl), 60 ~L ethanol, 1.12 mCi of IIInCl3 in O.OSN HCl
(80 ~,L)
and 155 ~.L sodium acetate buffer (0.5M, pH 4.5) at 85°C for 30 min.
Free' IIIn was
scavenged by adding 20 ~L of a 1 % Na2EDTA.2H20 (Aldrich) solution in water.
The
resulting radiochemical purity (RCP) was 87%. The radiolabeled product was
separated from
unlabeled ligand and other impurities by HPLC, using a Vydac Cl 8 column, [4.6
x 250 mm],
a column temperature of 50°C and a flow rate of 1.5 mL/min. with a
gradient of 75%A/25%B
to 65%A/35%B over 20 min where A is 0.1 % TFA in water, B is 0.085% TFA in
acetonitrile.
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With this system, the retention time for "'In-L64 is 15.7 min. The isolated
compound had an
RCP of 96.7%.
Example XLVIII - Preparation of ~~~Lu-DO3A-monoamide-Aoc-OWAVGHLM-NH2
Control
[00385] A stock solution of peptide was prepared by dissolving D03A-monoamide-
Aoc-
QWAVGHLM-NH2 ligand (prepared as described in US Application Publication No.
2002/0054855 and WO 02/87637, both incorporated by reference) in 0.01 N HCl to
a
concentration of 1 mg/mL. ~~~Lu- D03A-monoamide-Aoc-QWAVGHLM-NH2 was prepared
by mixing the following reagents in the order shown.
0.2 M NH40Ac, pH 6.8 100 ~L
Peptide stock, 1 mg/mL, in 0.01 N HCl 5 ~L
~~~LuCl3 (MURR) in 0.05M HCl 1.2 ~,L (1.4 mCi)
[00386] The reaction mixture was incubated at 85 °C for 10 min. After
cooling down to
room temperature in a water bath, 20 ~L of a 1 % EDTA solution and 20 ~L of
EtOH were
added. The compound was analyzed by HPLC using a C18 column (VYDAC Cat #
218TP54) that was eluted at flow rate of 1 mL/min with a gradient of 21 to 25%
B over 20
min, where A is 0.1 %TFA/H20 and B is 0.1 %TFA/CH3CN). ' ~~Lu--D03A-monoamide-
Aoc-QWAVGHLM-NH2 was formed in 97.1 % yield (RCP) and had a retention time of
16.1
min on this system.
Example XLIX - Preparation of l~~Lu-L63
[00387] This compound was prepared as described for l~~Lu- D03A-monoamide-Aoc-
QWAVGHLM-NH2. The compound was analyzed by HPLC using a C18 column (VYDAC
Cat # 218TP54) that was eluted at flow rate of 1 mL/min with a gradient of 30-
34% B over
20 min (where solvent is A. 0.1 %TFA/H20 and B is 0.1 %TFA/CH3CN). The'~~Lu-
L63 that
formed had an RCP of 97.8% and a retention time of 14.2 min on this system.
Example L - Preparation of ~~~Lu-L70 for cell binding and biodistribution
studies:
[00388] This compound was prepared following the procedures described above,
but
substituting L70 (the ligand of Example II). Purification was performed using
a YMC Basic
C8 column (4.6 x 150 mm), a column temperature of 30°C and a flow rate
of 1 mL/min. with
a gradient of 80%A/20%B to 75%A/25%B over 40 min., where A is citrate buffer
(0.02M,
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pH 4.5), and B is 80% CH3CN/20% CH30H. The isolated compound had an RCP of
100%
and an HPLC retention time of 25.4 min.
Example LI - Preparation of ~~~Lu- L70 for radiotherapy studies:
[00389] This compound was prepared as described above for L64.
Example LII - Preparation of ~~lIn-L70 for cell binding and biodistribution
studies:
[00390] This compound was synthesized by incubating 10 ~g L70 ligand (10 ~L of
a 1
m~mL solution in 0.01 N HCI), 180 ~L ammonium acetate buffer (0.2M, pH 5.3),
1.1 mCi
of "'InCl3 in O.OSN HCl (61 pL, Mallinckrodt) and 50 ~L of saline at
85°C for 30 min. Free
"'In was scavenged by adding 20 ~.L of a 1 % Na2EDTA~2H20 (Aldrich) solution
in water.
The resulting radiochemical purity (RCP) was 86%. The radiolabeled product was
separated
from unlabeled ligand and other impurities by HPLC, using a Waters XTerra C18
cartridge
linked to a Vydac strong anion exchange column [7.5 x 50 mm), a column
temperature of
30°C and a flow rate of 1 mL/min. with the gradient listed in the Table
below, where A is 0.1
mM NaOH in water, pH 10.0, B is 1 g/L ammonium acetate in water, pH 6.7 and C
is
acetonitrile. With this system, the retention time for "'In-L70 is 15 min
while the retention
time for L70 ligand is 27 to 28 min. The isolated compound had an RCP of 96%.
[00391 ] Samples for biodistribution and cell binding studies were prepared by
collecting
the desired HPLC peak into 500 ~.L of citrate buffer (0.05 M, pH 5.3,
containing 5% ascorbic
acid, 1 mg/mL L-methionine and 0.2% HSA). The organic part of the collection
was
removed by spin vacuum for 30 min. For cell binding studies, the purified,
concentrated
sample was used within 30 minutes of the in vitro study. For biodistribution
studies, the
sample was diluted with citrate buffer (0.05 M, pH 5.3, containing 5% sodium
ascorbic acid
and 0.2% HSA) to a final concentration of 10 ~Ci/mL within 30 minutes of the
in vivo study.
Time, min A B C
0-10 100%
10-11 100-50% 0-50%
11-21 50% 50%
21-22 50-0% 0-50% 50%
22-32 50% 50%
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Example LIII - IsZ vivo Pharmacokinetic Studies
A. Tracer dose biodistribution:
Low dose pharmacokinetic studies (e.g., biodistribution studies) were
performed using the below-identified compounds of the invention in
xenografted, PC3 tumor-bearing nude mice ([Ncr]-Foxnl <nu>). In all studies,
mice were administered 100 ~L of ~~~Lu-labeled test compound at 200 ~Ci/kg,
i.v., with a residence time of 1 and 24 h per goup (n=3-4). Tissues were
analyzed in an LKB 1282 CompuGamma counter with appropriate standards.
TABLE 7
[00392] Pharmacokinetic comparison at l and 24 h in PC3 tumor-bearing nude
mice
(200 ~.Cilkg; values as % ID/g) of ~~~Lu-177 labeled compounds of this
invention
compared to control
D03A-
monoamide-
Aoc-
QWAVGHL
M-NHZ
Tissue control L63 L64 L70
1 hr 24 1 hr 24 1 hr 24 1 24
hr hr hr hr hr
Blood 0.44 0.03 7.54 0.05 1.87 0.02 0.33 0.03
Liver 0.38 0.04 12.15 0.20 2.89 0.21 0.77 0.10
Kidneys 7.65 1.03 7.22 0.84 10.95 1.45 6.01 2.31
Tumor 3.66 1.52 9.49 2.27 9.83 3.60 6.42 3.50
Pancreas28.60 1.01 54.04 1.62 77.78 6.56 42.3440.24
[00393] Whereas the distribution of radioactivity in the blood, liver and
kidneys after
injection of L64 and L70 is similar to that of the control compound, DO3A-
monoamide-Aoc-
QWAVGHLM-NH2), the uptake in the tumor is much higher at 1 and 24 h for both
L64 and
L70. L63 also shows high tumour uptake although with increased blood and liver
values at
early times. Uptake in the mouse pancreas, a normal organ known to have GRP
receptors is
much higher for L64, L70 and L63 than for the control compound D03A-monoamide-
Aoc-
QWAVGHLM-NH2.
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Example LIV-Receptor Subtype Specificity
[00394] Currently, four mammalian members of the GRP receptor family are
known: the
GRP-prefernng receptor (GRP-R), neuromedin-B preferring receptor (NMB-R), the
bombesin receptor subtype 3 (BB3-R) and the bombesin receptor subtype 4 (BB4-
R). The
receptor subtype specificity of ~~~Lu-L70 was investigated. The results
indicate ~~~Lu-L70
binds specifically to GRP-R and NMB-R, and has little affinity for BB3-R.
[00395] The subtype specificity of the Lutetium complex of L70 (here, l~~Lu-
L70)
(prepared as described sutara) was determined by in vitro receptor
autoradiography using the
procedure described in Reubi et al., "Bombesin Receptor Subtypes in Human
Cancers:
Detection with the Universal Radioligand IZSI-[D-Tyr6, beta-Ala, Phel3,
N1e14]", CIin.Cancer
Res. 8:1139-1146 (2002) and tissue samples that had been previously found to
express only
one subtype of GRP receptor, as well as non-neoplastic tissues including
normal pancreas and
colon, as well as chronic pancreatitis (shown below in Table 8a). Human ileal
carcinoid
tissue was used as a source for NMB-R, human prostate carcinoma for GRP-R and
human
bronchial carcinoid for BB3-R subtype receptors. For comparison, receptor
autoradiography
was also performed with other bombesin radioligands, such as ~ZSI-Tyr4-
bombesin or a
compound known as the Universal ligand, ~2sI-[DTyrb, (3A1a11, Phel3, Nlel4]-
BBN(6-14),
which binds to all three subsets of GRP-R, on adjacent tissue sections. For
further
discussion, see Fleischmann et al., "Bombesin Receptors in Distinct Tissue
Compartments of
Human Pancreatic Diseases," Lab. Invest. 80:1807-1817 (2000); Markwalder et
al., "Gastin-
Releasing Peptide Receptors in the Human Prostate: Relation to Neoplastic
Transformation,"
Cancer Res. 59:1152-1159 (1999); Gugger et al., "GRP Receptors in Non-
Neoplastic and
Neoplastic Human Breast," Am. J. Pathol. 155:2067-2076 (1999).
TABLE 8A
Detection of bombesin receptor subtypes in various human tissues using
different radioligands.
Receptor Receptor
autoradiography autoradiography
Tumor n using using
~~~Lu-L70 standard
BN
radioligands*
GRP-R NMB-R BB3 GRP-R NMB-R BB3
Mammary Ca 8 8/8 0/8 0/8 8/8 0/8 0/8
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Receptor Receptor
autoradiography autoradiography
Tumor n using using
~~~Lu-L70 standard
BN
radioligands*
GRP-R NMB-R BB3 GRP-R NMB-R BB3
Prostate Ca 4 4/4 0/4 0/4 4/4 0l4 0/4
Renal Ca 6 5/6 0/6 O/6 4/6 0/6 0/6
heal carcinoid8 O/8 8/8 0/8 0/8 8/8 0/8
Bronchial 6 2/6 0/6 0/6 2/6 (weak)0/6 6/6
(weak)
carcinoid
Colon Ca
- tumor 7 3/7 0/7 0/7 3/7 (weak)0/7 0/7
(weak)
- smooth 7 7/7 0/7 0/7 7/7 0/7 0/7
muscle
Pancreas Ca 4 0/4 0/4 0l4 0/4 0/4 0/4
Chronic 5 5/5 0/5 0/5 5/5 0/5 0/5
pancreatitis
(acini)
Human pancreas7 1/7 0/7 0/7 0/7 0/7 0/7
(weak)
(acini)
Mouse pancreas4 4/4 0/4 0/4 4/4 0l4 0/4
(acini)
* i2sl-[DTyr6, ~iAlal~, Phe~3, NIe~4]-BBN(6-I4) and lasl-Tyr4-BBN.
[00396] A seen from Table 8a, all GRP-R-expressing tumors such as prostatic,
mammary
and renal cell carcinomas, identified as such with established radioligands,
were also
visualized in vitro with l~~Lu-L70. Due to a better sensitivity, selected
tumors with low
levels of GRP-R could be identified with l~~Lu-L70, but not with l2sl-Tyr4-
BBN, as shown in
Table 8a. All NMB-R-expressing tumors identified with established radioligands
were also
visualized with l~~Lu-L70. Conversely, none of the BB3 tumors were detected
with i~~Lu-
L70. One should not make any conclusion on the natural incidence of the
receptor
expression in the various types of tumors listed in Table 8a, as the tested
cases were chosen
as receptor-positive in the majority of cases, with only a few selected
negative controls. The
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normal human pancreas is not labeled with l~~Lu-L70, whereas the mouse
pancreas is
strongly labeled under identical conditions. Although the normal pancreas is a
very rapidly
degradable tissue and one can never completely exclude degradation of protein,
including
receptors, factors suggesting that the human pancreas data are truly negative
include the
positive control of the mouse pancreas under similar condition and the
strongly labeled BB3
found in the islets of the respective human pancreas, which represent a
positive control for
the quality of the investigated human pancreas. Furthermore, the detection of
GRP-R in
pancreatic tissues that are pathologically altered (chronic pancreatitis)
indicate that GRP-R,
when present, can be identified under the chosen experimental conditions in
this tissue. In
fact, ~~~Lu-L70 identifies these GRP-R in chronic pancreatitis with greater
sensitivity than
~25I-Tyr4-BBN. While none of the pancreatic cancers had measurable amounts of
GRP-R, a
few colon carcinomas showed a low density of heterogeneously distributed GRP
receptors
measured with l~~Lu-L70 (Table 8a). It should further be noticed that the
smooth muscles of
the colon express GRP-R and were detected in vitro with l~~Lu-L70 as well as
with the
established bombesin ligands.
TABLE r3B
[00397] Binding affinity of 1~$Lu-L70 to the 3 bombesin receptor subtypes
expressed in
human cancers. Data are expressed as ICso in nM (mean ~ SEM. n = number of
experiments
in parentheses).
Compound B. NMB-R C. GRP-R BB3
Universal ligand0.8 0.1 (3) 0.7 0.1 (3) l .l 0.1 (3)
msLu-L70 0.9 0.1 (4) 0.8 0.1 (5)
>1,000 (3)
[00398] As shown in Table 8b, the cold labeled x~SLu-L70 had a very high
affinity for
human GRP and NMB receptors expressed in human tissues while it had only low
affinity for
BB3 receptors. These experiments used ~25I-[DTyr6, (3A1a11, Phe~3, Nle~4]-
BBN(6-14) as
radiotracer. Using the ~~~Lu-labeled L70 as radiotracer, the above mentioned
data are hereby
confirmed and extended. All GRP-R-expressing human cancers were very strongly
labeled
with ~~~Lu-L70. The same was true for all NMB-R-positive tumors. Conversely,
tumors
with BB3 were not visualized. The sensitivity of ~~~Lu-L70 seems better than
that of Iasl-
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Tyr4-BBN or the'25I-labeled universal bombesin analog. Therefore, a few tumors
expressing
a low density of GRP-R can be readily identified with'~~Lu-L70, while they are
not positive
with'ZSI-Tyr4-BBN. The binding characteristics of ~~~Lu-L70 could also be
confirmed in
non-neoplastic tissues. While the mouse pancreas, as control, was shown to
express a very
high density of GRP-R, the normal human pancreatic acini were devoid of GRP-R.
However, in conditions of chronic pancreatitis GRP-R could be identified in
acini, as
reported previously in Fleisclunann et al., "Bombesin Receptors in Distinct
Tissue
Compartments of Human Pancreatic Diseases," Lab. Invest. 80:1807-1817 (2000)
and tissue,
again with better sensitivity by using l~~Lu-L70 than by using'25I-Tyr4-BBN.
Conversely,
the BB3-expressing islets were not detected with l~~Lu-L70, while they were
strongly labeled
with the universal ligand, as reported previously in Fleischmann et al.,
"Bombesin Receptors
in Distinct Tissue Compartments of Human Pancreatic Diseases," Lab. Invest.
80:1807-1817
(2000). While a minority of colon carcinomas had GRP-R, usually in very low
density and
heterogeneously distributed, the normal colonic smooth muscles expressed a
high density of
GRP-R.
[00399] The results in Tables 8a and 8b indicate that Lu labeled L70
derivatives are
expected to bind well to human prostate carcinoma, which primarily expresses
GRP-R. They
also indicate that Lu labeled L70 derivatives are not expected to bind well to
normal human
pancreas (which primarily expresses the BB3-R receptor), or to cancers which
primarily
express the BB3-R receptor subtype.
Examine LV - Radiotherapy Studies
A. Efficac~Studies:
[00400) Radiotherapy studies were performed using the PC3 tumor-bearing nude
mouse model. In Short Term Efficacy Studies, '~~Lu labeled compounds of
the invention L64, L70, L63 and the treatment control compound D03A-
monoamide-Aoc-QWAVGHLM-NH2 were compared to an untreated control
group. (n=12 for each treatment group for up to 30 days, and n=36 for the
pooled untreated control group for up to 31 days). For all efficacy studies,
mice were administered 100 p.L of'~~Lu-labeled compound of the invention at
30 mCi/kg, i.v, or s.c. under sterile conditions. The subjects were housed in
a
barrier environment for the duration of the study. Body weight and tumor size
(by caliper measurement) were collected on each subject 3 times per week for
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the duration of the study. Criteria for early termination included: death;
loss
of total body weight (TBW) equal to or greater than 20%; tumor size equal to
or greater than 2 cm3. Results of the Short Term Efficacy Study are displayed
in FIG.15A. These results show that animals treated with L70, L64 or L63
have increased survival over the control animals given no treatment and over
those animals given the same dose of the D03A-monoamide-Aoc-
QWAVGHLM-NH2 control.
[00401] Long Term Efficacy Studies were performed with L64 and L70 using the
same dose as before but using more animals per compound (n=46) and
following them for up to 120 days. The results of the Long Term Efficacy
Study are displayed in FIG.15B. Relative to the same controls as before
(n=36), both L64 and L70 treatment gave significantly increased survival
(p<0.0001) with L70 being better than L64, although not statistically
different
from each other (p<0.067).
Example LVI
Alternative Preparation of L64 and L70 Using Segment Coupling
[00402] Compounds L64 and L70 can be prepared employing the collection of
intermediates generally represented by A-D (FIG.19), which themselves are
prepared by
standard methods known in the art of solid and solution phase peptide
synthesis (Synthetic
Peptides - A User's Guide 1992, Grant, G., Ed. WH. Freeman Co., NY, Chap 3 and
Chap 4
pp. 77 - 258; Chan, W.C. and White, P.D. Basic Procedures in Fmoc Solid Phase
Peptide
Synthesis - A Practical Approach 2002, Chan, W.C. and White, P.D. Eds Oxford
University
Press, New York, Chap. 3 pp 41 - 76; Barlos, K and Gatos, G. Convergent
Peptide Synthesis
in Fmoc Solid Phase Peptide Synthesis - A Practical Approach 2002, Chan, W.C.
and White,
P.D. Eds Oxford University Press, New York, Chap. 9 pp. 216 - 228) which are
incorporated
herein by reference.
[00403] These methods include Aloc, Boc, Fmoc or benzyloxycarbonyl-based
peptide
synthesis strategies or judiciously chosen combinations ofthose methods on
solid phase or in
solution. The intermediates to be employed for a given step are chosen based
on the selection
of appropriate protecting groups for each position in the molecule, which may
be selected
from the list of groups shown in FIG. 1. Those of ordinary skill in the art
will also
understand that intermediates, compatible with peptide synthesis methodology,
comprised of
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alternative protecting groups can also be employed and that the listed options
for protecting
groups shown above serves as illustrative and not inclusive, and that such
alternatives are
well known in the art.
[00404] This is amply illustrated in FIG. 20 which outlines the approach.
Substitution of
the intermediate C2 in place of C1 shown in the synthesis of L64, provides L70
when the
same synthetic strategies are applied.
EXAMPLE LVII - Figures 49A and 49B
Synthesis of L69
[00405] Summary: Reaction of (313,513,7a,12a)-3-amino-7,12-dihydroxycholan-24-
oic acid
A with Fmoc-Cl gave intermediate B. Rink amide resin functionalised with the
octapeptide
Gln-Trp-Ala-Val-Gly-His-Leu-Met-NHZ (BBN[7-14]) (A), was sequentially reacted
with B,
Fmoc-8-amino-3,6-dioxaoctanoic acid and DOTA tri-t-butyl ester. After cleavage
and
deprotection with Reagent B the crude was purified by preparative HPLC to give
L230.
Overall yield: 4.2%.
A. (3J3,SJ3,7a,12a)-3-(9H-Fluoren-9-ylmethoxy)amino-7,12-dihydroxycholan-24-
oic acid, B (FIG. 49A)
[00406] A solution of 9-fluorenylmethoxycarbonyl chloride (1.4 g; 5.4 mmol) in
1,4-
dioxane (18 mL) was added dropwise to a suspension of (313,513,7a,12a)-3-
amino-7,12-dihydroxycholan-24-oic acid A (2.0 g; 4.9 mmol) (3) in 10% aq.
Na2C03 (30 mL) and 1,4-dioxane (18 mL) stirred at 0 °C. After 6 h
stirring at
room temperature H20 (100 mL) was added, the aqueous phase washed with
Et20 (2 x 90 mL) and then 2 M HCl (15 mL) was added (final pH: 1.5). The
precipitated solid was filtered, washed with H20 (3 x 100 mL), vacuum dried
and then purified by flash chromatography to give B as a white solid (2.2 g;
3.5 mmol). Yield 71 %.
B. N-[313,513,7a,12a)-3-[[[2-[2-[[[4,7,10-Tris(carboxymethyl)-1,4,7,10-
tetraazacyclododec-1-yl]acetyl]amino] ethoxy]ethoxy] acetyl]amino]-7,12-
dihydroxy-24-oxocholan-24-yl]-L-glutaminyl-L-tryptophyl-L-alanyl-L-valyl-
g-l~yl-L-histidyl-L-leucyl-L-methioninamide, L69 (FIG. 49B1
[00407] Resin A (0.5 g; 0.3 mmol) was shaken in a solid phase peptide
synthesis vessel
with 50% morpholine in DMA (7 mL) for 10 min, the solution filtered and
fresh 50% morpholine in DMA (7 mL) was added. The suspension was stirred
for another 20 min then the solution was filtered and the resin washed with
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DMA (5 x 7 mL). (313,513,7a,12a)-3-(9H-Fluoren-9-ylmethoxy)amino-7,12-
dihydroxycholan-24-oic acid B (0.75 g; 1.2 mmol), N-hydroxybenzotriazole
(HOBt) (0.18 g; 1.2 mmol), N,N'-diisopropylcarbodiimide (DIC) (0.19 mL;
1.2 mmol) and DMA (7 mL) were added to the resin, the mixture shaken for
24 h at room temperature, emptied and the resin washed with DMA (5 x 7
mL). The resin was then shaken with 50% morpholine in DMA (7 mL) for 10
min, the solution emptied, fresh 50% morpholine in DMA (7 mL) was added
and the mixture shaken for another 20 min. The solution was emptied and the
resin washed with DMA (5 x 7 mL). Fmoc-8-amino-3,6-dioxaoctanoic acid
(0.79 g; 1.2 mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL; 1.2 mmol) and
DMA (7 mL) were added to the resin. The mixture was shaken for 3 h at
room temperature, emptied and the resin washed with DMA (5 x 7 mL). The
resin was then shaken with 50% morpholine in DMA (7 mL) for 10 min, the
solution filtered, fresh 50% morpholine in DMA (7 mL) was added and the
mixture shaken for another 20 min. The solution was filtered and the resin
washed with DMA (5 x 7 mL) 1,4,7,10-Tetraazacyclododecane-1,4,7,10-
tetraacetic acid tris(1,1-dimethylethyl) ester adduct with NaCI (0.79 g; 1.2
mmol), HOBt (0.18 g; 1.2 mmol), DIC (0.19 mL: 1.2 mmol), N-
ethyldiisopropylamine (0.40 mL; 2.4 mmol) and DMA (7 mL) were added to
the resin. The mixture was shaken for 24 h at room temperature, filtered and
the resin washed with DMA (5 x 7 mL), CH2Cl2 (5 x 7 mL) and vacuum dried.
The resin was shaken in a flask with Reagent B (25 mL) (2) for 4.5 h. The
resin was filtered and the solution was evaporated under reduced pressure to
afford an oily crude that after treatment with EtzO (20 mL) gave a
precipitate.
The precipitate was collected by centrifugation and washed with Et20 (3 x 20
mL) to give a solid (248 mg) which was analysed by HPLC. An amount of
crude (50 mg) was purified by preparative HPLC. The fractions containing
the product were lyophilised to give L69 (6.5 mg; 3.5 x 10-3 mmol) (FIG.
49B) as a white solid. Yield 5.8%.
EXAMPLE LVIII - Figure 50
Synthesis of L144
[00408] Summary: Rink amide resin functionalised with the octapeptide Gln-Trp-
Ala-
Val-Gly-His-Leu-Met-NHZ (BBN[7-14]) (A) was reacted with 4-[2-hydroxy-3-
[4,7,10-tris[2-
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(1,1-dimethylethoxy)-2-oxoethylJ-1,4,7,10-tetrazacyclododec-1-yl)propoxy]
benzoic acid.
After cleavage and deprotection with Reagent B (2) the crude was purified by
preparative
HPLC to give L144. Overall yield: 12%.
A. N [4-[2-Hydroxy-3-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec
1-yl]propoxy]benzoyl]-L-glutaminyl-L-tryptophyl-L-al anyl-L-valyl-glycyl-L
histidyl-L-leucyl-L-methioninamide, L144 (FIG. 50)
[00409] Resin A (0.4 g; 0.24 mmol) was shaken in a solid phase peptide
synthesis
vessel with 50% morpholine in DMA (7 mL) for 10 min, the solution filtered
and fresh 50% morpholine in DMA (7 mL) was added. The suspension was
stirred for another 20 min then the solution was filtered and the resin washed
with DMA (5 x 7 mL). 4-[2-Hydroxy-3-[4,7,10-tris[2-(1,1-dimethylethoxy)-
2-oxoethyl]-1,4,7,10-tetrazacyclododec-1-yl]propoxy] benzoic acid B (0.5 g;
0.7 mmol), HOBt (0.11 g; 0.7 mmol), DIC (0.11 mL; 0.7 mmol)), N
ethyldiisopropylamine (0.24 mL; 1.4 mmol) and DMA (7 mL) were added to
the resin. The mixture was shaken for 24 h at room temperature, emptied and
the resin washed with DMA (5 x 7 mL), CHZC12 (5 x 7 mL) and vacuum dried.
The resin was shaken in a flask with Reagent B (25 mL) (2) for 4.5 h. The
resin was filtered and the solution was evaporated under reduced pressure to
afford an oily crude that after treatment with Et20 (20 mL) gave a
precipitate.
The precipitate was collected by centrifugation and washed with Et20 (3 x 20
mL) to give a solid (240 mg) which was analysed by HPLC. An amount of
crude (60 mg) was purified by preparative HPLC. The fractions containing
the product were lyophilised to give L144 (10.5 mg; 7.2 x 10-3 mmol) as a
white solid. Yield 12%.
EXAMPLE LIX
Preparation of L300 and ~~~Lu-L300
[00410] From 0.2 g of Rink amide Novagel resin (0.63 mmol/g, 0.126 mmol), L300
(0.033
g, 17%) was obtained after preparative column chromatography. The retention
time was 6.66
minutes. The molecular formula is C~aH99N190~s. The calculated molecular
weight is
151 x.71; 1519.6 observed. The sequence is D03A-Gly-Abz4-Gln-Trp-Ala-Val-Gly-
His-
Phe-Leu-NH2. The structure of L300 is shown in Figure S1.
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[00411 ] L300 (13.9 ~g in 13.9 ~L of 0.2M pH 4.8 sodium acetate buffer) was
mixed with
150 ~L of 0.2M pH 4.8 sodium acetate buffer and 4 ~L of ~~~LuCl3 (1.136 mCi,
Missouri
Research Reactor). After 10 min at 100°C, the radiochemical purity
(RCP) was 95%. The
product was purified on a Vydac C18 peptide colunm (4.6 x 250 mm, 5 um pore
size) eluted
at a flow rate of 1 mL/min using an aqueous/organic gradient of 0.1 % TFA in
water (A) and
0.085% TFA in acetonitrile (B). The following gradient was used: isocratic 22%
B for 30
min, to 60% B in 5 min, hold at 60% B for 5 min. The compound, which eluted at
a retention
time of 18.8 min., was collected into 1 mL of an 0.8% human serum albumin
solution that
was prepared by adding HSA to a 9:1 mixture of normal saline and Ascorbic
Acid, Injection.
Acetonitrile was removed using a Speed Vacuum (Savant). After purification,
the compound
had an RCP of 100%.
EXAMPLE LX - Characterization of Linker Specificity in Relation to GRP
Receptor
Subtypes
[00412] Two cell lines, C6, an NMB-R expressing rodent glioblastoma cell line
and PC3,
a GRP-R expressing human prostate cancer cell line, were used in this assay.
The affnity of
various unlabeled compounds for each receptor subtype (NMB-R and GRP-R) was
determined indirectly by measuring its ability to compete with the binding of
~ZSI-NMB or
izsl-BBN to its corresponding receptors in C6 and PC3 cells.
A. Materials and Methods:
1. Cell Culture:
[00413] C6 cells were obtained from ATCC (CCL-107) and cultured in F12K media
(ATCC) supplemented with 2 mM L-glutamine, 1.5 g/L Sodium bicarbonate,
15% horse serum and 2.5% FBS. Cells for the assays were plated at a
concentration of 9.6 x 104/ well in 48 well poly-lysine coated plates (Beckton
Dickinson Biocoat). PC3 were obtained from ATCC (CRL-1435) and
cultured in RPMI 1640 (ATCC) supplemented with 2 mM L-glutamine, 1.5
g/L Sodium bicarbonate, 10 mM HEPES and 10% FBS. Both cultures were
maintained in a humidified atmosphere containing 5% C02/95% air at
37°C.
PC3 cells for the assays were plated at a concentration of 2.0 x 104 cells/
well
in 96-well white/clear bottom plates (Falcon Optilux-I). Plates were used for
the assays on day 2 of the post-plating.
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2. Binding buffer, and radio-li ands:
[00414] RPMI 1640 (ATCC) containing 25 mM HEPES, 0.2% BSA fraction V, 1.0
mMAEBSF (CAS # 3087-99-7) and 0.1 % Bacitracin (CAS # 1405-87-4), pH
7.4.
Custom made ~2sI-[Tyr°]NMB, > 2.0 Ci/pmole (Amersham Life
Science) [~2sI-NMB] and commercially available ~ZSI-[Tyr4]BBN, >2.0
Cilpmole (Perkin Elmer Life Science) [l2sl-BBN] were used as
radioligands.
B. In vitro Assay:
[00415] Using a 48-well plate assay system (for C6 study) competition
experiments
were performed using ~ZSI-NMB. All of the PC3 studies were performed as
described in Example ~LIII using ~2sI-BBN. Selection of compounds for the
assay was based on linker subtype. Results are shown in Table 9.
TABLE 9
Number of selected compounds for the assay and their linkers
biphenyl)
[00416] The binding parameters obtained from the studies were analyzed using a
one-site
competition non-linear regression analysis with GraphPad Prism. The relative
affinity of
various compounds for NMB-R in C6 cells were compared with those obtained
using
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commercially available [Tyr4]-BBN and [Tyr°]-NMB. To distinguish the
GRP-R preferring
compounds from NMB-R plus GRP-R prefernng compounds, ICs° values
obtained for each
compound was compared with those obtained from [Tyr°]-BBN with ~zsI-NMB
on C6 cells.
The cut off point between the two classes of compounds was taken as 1 OX the
ICs° of [Tyr4]-
BBN. Among the compounds tested, 8 compounds preferentially bind to GRP-R (as
shown
in Table 10) while 32 compounds bind to both GRP-R and NMB-R with similar
affinity, and
two show preference for NMB-R.
TABLE 10
The ICS° values obtained from competition experiments using 125I-NMB
and 125I-BBN
L # COMPOUND ICSO (nM) GRP-R GRP-R
~zsl-BBN 'zsI-NMB
lPC3 !C6 NMB-R
na N,N-dimethylglycine-Ser-10 10.4 - yes
Cys(Acm)-Gly-SS-
WAVGHLM-NHz
na N,N-dimethylglycine-Ser-25 7.9 - yes
Cys(Acm)-Gly -G-
WAVGHLM-NHz
na N,N-dimethylglycine-Ser-48 20.2 - yes
Cys(Acm)-Gly -GG-
WAVGHLM-NHz
na N,N-dimethylglycine-Ser-13 6.4 - yes
Cys(Acm)-Gly -KK-
WAVGHLM-NHz
na N,N-dimethylglycine-Ser-2 2.2 - yes
Cys(Acm)-Gly -SK-
WAVGHLM NHz
na N,N-dimethylglycine-Ser-1.9 2.0 yes
Cys(Acm)-Gly -SR- -
WAV GHLM-NH2
na N,N-dimethylglycine-Ser-7.5 24.1
Cys(Acm)-Gly -KS- yes -
WAVGHLM-NHz
na N,N-dimethylglycine-Ser-32 60.0
Cys(Acm)-Gly -KE- yes -
WAVGHLM-NHz
na D03A-monoamide-Aoc- 3.4 3.1 yes
WAVGHLM-NH2 _
na D03A-monoamide -Apa3- 36 18. 9 yes
WAVGHLM-NHz _
na D03A-monoamide Abu4- 19.8 5.2 yes
WAVGHLM-NHz _
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J., # COMPOUND ICso (nM) GRP-R GRP-R
~zsl-BBN 'ZSI-NMB &
/PC3 1C6 NMB-R
L3 N,N-dimethylglycine-Ser-70 33 yes
Cys(Acm)-Gly - DJ- -
WAVGHLM-NHZ
L64 DO3A-monoamide -G- 8.5 3.3 yes
Adca3- WAVGHLM-NH2 _
L63 DO3A-monoamide -G- 23 3.8 yes
Ahl2ca- WAVGHLM-NH2 _
L67 DO3A-monoamide -G-Akca-5.5 2.3 yes
WAVGHLM- NHZ _
na D03A-monoamide -Cha- 22 77 -
Cha- WAVGHLM- NH2 yes
na D03A-monoamide -Nal1- 30 210.9 -
Bi - WAVGHLM- NH2
yes
na DO3A-monoamide -Cha- 8 66.5 -
Na11- WAVGHLM- NH2
yes
na D03A-monoamide -Nall- 17 89.9 -
B a4- WAVGHLM- NH2
yes
L301 D03A-monoamide -Amb4- 10 6.8
yes
Nall- WAVGHLM- NH2
L147 D03A-monoamide -G- 4 32
Mo3abz4-QWAVGHLM- yes -
NH2
L24I D03A-monoamide -G- 4 0.8
Cl3abz4 WAVGHLM- NH2 - yes
L242 D03A-monoamide -G- 5 2.2
M3abz4-QWAVGHLM- - yes
NH2
L243 DO3A-monoamide -G- 14 9.9
Ho3abz4-QWAVGHLM- - yes
NH2
L202 D03A-monoamide -G- 13 2.7
- yes
H bz4- WAVGHLM- NH2
L204 D03A-monoamide -Abz4- 50 1.2
- yes
G- WAVGHLM- NH2
L233 D03A-monoamide -G- 4.8 1.6
- yes
Abz3- WAVGHLM- NHZ
L235 D03A-monoamide -G- 7 1.5
Nmabz4-QWAVGHLM- - yes
NHa
L147 D03A-monoamide - 3.5 1.2
Mo3amb4-QWAVGHLM- - yes
NH2
L71 D03A-monoamide -Amb4- 7.2 0. 2
- yes
WAVGHLM- NHa
L73 D03A-monoamide -Aeb4- 5 1.8
- yes
WAVGHLM- NH2
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I~ # COMPOUND ICso (nM) GRP-R GRP-R
izsl_BBN 'ZSI-NMB &
/PC3 /C6 NMB-R
L208 D03A-monoamide -Dae- 8 0.9
- yes
T a- WAVGHLM- NHZ
L206 D03A-monoamide -G- 5 1.3
A4m2biphc4- - yes
WAVGHLM- NHZ
L.207 D03A-monoamide -G- 3 15.1
A3biphc3-QWAVGHLM- - yes
NH2
L72 D03A-monoamide -Amc4-8.2 2.6
- yes
WAVGHLM- NH2
L107 D03A-monoamide -Amc4-5 0.3
- yes
Amc4- WAVGHLM- NH2
L89 D03A-monoamide -Aepa4-23 114
yes -
WAVGHLM- NH2
L28 N,N-dimethylglycine-Ser-25 13
Cys(Acm)-Gly - Aepa4-S- - yes
WAVGHLM- NH2
L74 D03A-monoamide -G-Inp-6.5 3.4
- yes
WAVGHLM- NH2
L36 N,N-dimethylglycine-Ser-7 12.1
Cys(Acm)-Gly - Pial-J- - yes
WAVGHLM- NH2
L82 D03A-monoamide -Ckbp-8 1.7 -
yes
WAVGHLM- NH2
na D03A-monoamide -Aoc- 11 14 -
yes
WAVGHL-Nle-NH2
L70 D03A-monoamide -G- 4.5 1.5 -
yes
Abz4- WAVGHLM- NHz
na D03A-monoamide - 366 >250 No selective
WAVGHLM- NHS reference
na QWAVGHLM- NHZ 369 754 No selective
reference
na WAVGHLM -NHS >800 >800 No selective
reference
L204 DO3A-monoamide -Abz4->50 1.2 preference
to NMB-
G- WAVGHLM- NH2 R
na GNLWATGHFM-NH2 >500 0.7 preference
to NMB-
R
L227 D03A-monoamide -G- 28 0.8 - Ye
Abz4-LWATGHFM -NHa s
In the above Table "na" indicates "not applicable" (e.g., the compound does
not contain a
linker of the invention and thus was not assigned an L#).
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[00417] Based on the above, several results were observed. The receptor
binding region
alone (BBN~_~4 or BBN$_~4) did not show any preference to GRP-R or NMB-R. The
addition
of a chelator alone to the receptor binding region did not contribute to the
affinity of the
peptide to GRP-R or NMB-R (D03A-monoamide -QWAVGHLM-NH2). Coupling the
chelator to the peptide through a linker did contribute to the affinity of the
peptide towards
the receptor. However, depending on the type of linker this affinity varied
from being dual
(preference for both NMB-R and GRP-R) to GRP-R (preferring GRP-R).
[00418] The cn-Aminoalkanoic acids tested (8-Aminooctanoic acid in ~~SLu- D03A-
monoamide-Aoc-QWAVGHLM-NH2 and D03A-monoamide -Aoc-QWAVGHL-Nle-NH2 , 3-
aminopropionic acid in D03A-monoamide -Apa3-QWAVGHLM-NH2 and 4-aminobutanoic
acid in D03A-monoamide-Abu4-QWAVGHLM-NH2) as linkers, conferred the peptide
with dual affinity for both GRP-R and NMB-R. Replacement of 'Met' in I~SLu-
DOTA-Aoc-
QWAVGHLM NH2 by 'Nle' did not change this dual affinity of the peptide.
[00419] Cholic acid containing linkers (3-aminocholic acid in L64, 3-amino-12-
hydroxycholanic in L63 and 3-amino-12-ketocholanic in L67 conferred the
peptides with
dual affinity for both GRP-R and NMB-R. Cycloalkyl and aromatic substituted
alanine
containing linkers (3-cyclohexylalanine in DO3A-monoamide -Cha-Cha-QWAVGHLM-
NH2, 1-Naphthylalanine in D03A-monoamide-Cha-Nal 1-QWAVGHLM-NH2, 4-
Benzoylphenylalanine in DO3A-monoamide -Nall-Bpa4-QWAVGHLM-NH2 and
Biphenylalanine in DO3A-monoamide -Nall-Bip-QWAVGHLM-NHZ) imparted the
peptides
with selective affinity towards GRP-R. A linker containing only 4-(2-
Aminoethylpiperazine)-1 also contributed to the peptides with GRP-R
selectivity (L89).
[00420] Introduction of G-4-amino benzoic acid linker to NMB sequence
conferred the
compound with an affinity to GRP-R in addition to its inherent NMB-R affinity
(L227 vs
GNLWATGHFM-NHz). Shifting the position of Gly around the linker altered the
affinity of
L70 from its dual affinity to a selective affinity to NMB-R (L204). 3-methoxy
substitution in
4-aminobenzoic acid in L70 (as in L240) changed the dual affinity to a
selective affinity to
GRP-R.
[00421 ] It is apparent from the preceding data that the linker has a
significant effect on the
receptor subtype specificity. Three groups of compounds can be identified:
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~:~ Those that are active at the GRP-R
[00422] These compounds provide information specific to this receptor in vitro
and in
vivo, which can be used for diagnostic purposes. When these compounds are
radiolabeled with a therapeutic radionuclide, therapy can be performed on
tissues containing only this receptor, sparing those that contain the NMB-R
~:~ Those that are active at the NMB-R
[00423] These compounds provide information specific to this receptor in vitro
and in
vivo, which can be used for diagnostic purposes. When radiolabeled with a
therapeutic radionuclide, therapy can be performed on tissues containing only
this receptor, sparing those that contain the GRP-R
~:~ Those that are active at both the GRP-R and the NMB-R
[00424] These compounds provide information on the combined presence of these
two
receptor subtypes in vitro and in vivo, that can be used for diagnostic
purposes. Targeting both receptors may increase the sensitivity of the
examination at the expense of specificity. When these compounds are
radiolabeled with a therapeutic radionuclide, therapy can be performed on
tissues containing both receptors, which may increase the dose delivered to
the
desired tissues.
Example LXI - Competition studies of modified Bombesin (BBN) analogs with lzsl-
BBN
for GRP-R in human prostate cancer (PC3) cells
[00425] To determine the effect of replacing certain amino acids in the BBN ~-
~4 analogs,
peptides modified in the targeting portion were made and assayed for
competitive binding to
GRP-R in human prostate cancer (PC3) cells. All these peptides have a common
linker
conjugated to a metal chelating moiety (DOTA-Gly-Abz4-). The binding data
(ICSO nM) are
given below in Table 13.
A. Materials and Methods:
1. Cell Culture:
[00426] PC3 cell lines were obtained from ATCC (CRL-1435) and cultured in RPMI
1640 (ATCC) supplemented with 2 mM L-glutamine, 1.5 glL Sodium
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bicarbonate, 10 mM HEPES and 10% FBS. Cultures were maintained in a
humidified atmosphere containing 5% C02/95% air at 37 °C. PC3 cells for
the
assays were plated at a concentration of 2.0 x 104 cells/ well in a 96-well
white/clear bottom plates (Falcon Optilux-I). Plates were used for the assays
on day 2 of the post-plating.
2. Binding buffer:
[00427] RPMI 1640 (ATCC) containing 25 mM HEPES, 0.2% BSA fraction V, 1.0
mM AEBSF (CAS # 3087-99-7) and 0.1 % Bacitracin (CAS # 1405-87-4), pH
7.4.
3. ~2sI-Tyr4-Bombesin [~ZSI-BBNI
[00428] ~2sI-BBN (Cat # NEX258) was obtained from PerkinElmer Life
Sciences.
C. In vitro Assay:
[00429] Competition assay with ~ZSI-BBN for GRP-R in PC3 cells:
[00430] All compounds tested were dissolved in binding buffer and appropriate
dilutions were also done in binding buffer. PC3 cells for assay were seeded at
a concentration of 2.0 x 104 /well either in 96-well black/clear collagen I
cellware plates (Beckton Dickinson Biocoat). Plates were used for binding
studies on day 2 post- plating. The plates were checked for confluency (>90%
confluent) prior to assay. For competition assay, N,N-dimethylglycyl-Ser-
Cys(Acm)-Gly-AvaS-QWAVGHLM-NH2 (control) or other competitors at
concentrations ranging from 1.25x 10-9 M to 500 x 109 M, was co-incubated
with lasl-BBN (25,000 cpmlwell). The studies were conducted with an assay
volume of 75 ~l per well. Triplicate wells were used for each data point.
After the addition of the appropriate solutions, plates were incubated for 1
hour at 4°C. Incubation was ended by the addition of 200 uL of ice-cold
incubation buffer. Plates were washed 5 times and blotted dry. Radioactivity
was detected using either a LIMB CompuGamma counter or a microplate
scintillation counter. The bound radioactivity of ~2sI-BBN was plotted against
the inhibition concentrations of the competitors, and the concentration at
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which l2sl-BBN binding was inhibited by 50% (ICso) was obtained from the
binding curve.
TABLE 13: Competition studies with lzsl-BBN for GRP-R in PC3 cells
ICso
L # PEPTIDES [nM]
N,N-dimethylglycyl-Ser-Cys(Acm)-Gly-AvaS-
Ref na QWAVGHLM-NHZ 2.5
1 L70 D03A-monoamide-G-Abz4-QWAVGHLM-NHZ 4.5
2 L214 D03A-monoamide-G-Abz4-fQWAVGHLM-NHZ 18
3 L215 D03A-monoamide-G-Abz4-QRLGNQWAVGHLM-NH2 6
4 L216 D03A-monoamide-G-Abz4-QRYGNQWAVGHLM-NH2 4.5
L217 D03A-monoamide-G-Abz4-QKYGNQWAVGHLM-NHZ 10
>EQ-[K(D03A-monoamide-G-Abz4)-LGNQWAVGHLM-
6 L218 NH2 53
7 L219 D03A-monoamide-G-Abz4-fQWAVGHLM-NH-CSH~Z 75
8 L220 DO3A-monoamide-G-Abz4-QWAVaHLM-NH2 13
9 L221 D03A-monoamide-G-Abz4-fQWAVGHLL-NHZ 340
L222 DO3A-monoamide-G-Abz4-yQWAV-Ala2-HF-Nle-NH246
11 L223 D03A-monoamide-G-Abz4-FQWAV-Ala2-HF-Nle-NH252
12 L224 D03A-monoamide-G-Abz4-QWAGHFL-NH2 >500
13 L225 D03A-monoamide-G-Abz4-LWAVGSFM-NH2 240
14 L226 D03A-monoamide-G-Abz4-HWAVGHLM-NH2 5.5
L227 D03A-monoamide-G-Abz4-LWATGHFM-NH2 39
16 L228 D03A-monoamide-G-Abz4-QWAVGHFM-NHZ 5.5
17 na GNLWATGHFM-NHZ >500
18 na yGNLWATGHFM-NH2 450
19 L300 D03A-monoamide-G-Abz4-QWAVGHFL-NH2 2.5
5 [00431 ] Results/Conclusions: Analysis of the binding results of various
peptides modified
in the targeting portion indicated the following:
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[00432] Neuromedin analogs (GNLWATGHFM-NHZ, yGNLWATGHFM-NH2) are unable
to compete for the GRP-R except when conjugated to D03A-monoamide-G-Abz4
(L227).
They are, however, effective NMB competitors. This is similar to the
requirement for
derivatisation of the amino end of the bombesin sequence as reflected in
QWAVGHLM-NHZ,
D03A-monoamide-QWAVGHLM-NHS & L70. Replacement of the histidine (L225)
reduces competition at the GRP-R.
[00433] Reversal of the two linker components in L70 to give L204 changes the
subtype
specificity to favor the NMB subtype. L~3F substitution in the bombesin
sequence maintains
GRP-R activity. (L228).
TABLE 14
ICso
L Number Sequence C6/NMB- PC3/GRP
R -R
na GNLWATGHFM-NHZ 0.69 >500
na yGNLWATGHFM-NH2 0.16 884.6
L227 D03A-monoamide-G-Abz4-LWATGHFM-NH2 0.07 28.0
L225 D03A-monoamide-G-Abz4-LWAVGSFM-NH2 - 240
na WAVGHLM-NH2 >800 >800
na QWAVGHLM-NHZ 369 754
na D03A-monoamide-QWAVGHLM-NHZ 161 366
L70 D03A-monoamide-G-Abz4-QWAVGHLM-NH2 4.5 1.5
L204 D03A-monoamide-Abz4-GQWAVGHLM-NH2 1.19 >50
L228 ~ D03A-monoamide-G-Abz4-QWAVGHFM-NH2 ~ 5.5
~
[00434] As seen here, Fl3Mia to F13L14 substitution in L228 produces a
compound (L300)
with high activity at the GRP-R. The removal of the methionine has advantages
as it is prone
to oxidation. This benefit does not occur if the L13F substitution is not also
performed
1 S (L221) . Removal of V 1° resulted in complete loss of binding as
seen in L224.
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TABLE 15
IC50
Number Sequence C6/NMB- PC3/GRP-
R R
L300 D03A-monoamide-G-Abz4-QWAVGHFL-NHZ - 2.5
L221 D03A-monoamide--G-Abz4-f WAVGHLL-NH2- 340
L224 D03A-monoamide-G-Abz4-QWA GHFL-NH2 - >500
TABLE 16
As seen in Table 16, various substitutions are allowed in the BBN2-6 region
(L214-L217,
L226)
Number Sequence IC50
C6/NMB- PC3/GRP-
R R
pEQRYGNQWAVGHLM-NH2 3.36 2.2
na
L214 D03A-monoamide-G-Abz4-fQWAVGHLM-NHZ - 18
D03A-monoamide-G-Abz4-
L215 QRLGNQWAVGHLM-NH2 - 6
D03A-monoamide-G-Abz4-
L216 QRYGNQWAVGHLM-NH2 - 4.5
D03A-monoamide-G-Abz4-
L217 QKYGNQWAVGHLM-NHa - 10
L226 D03A-monoamide-G-Abz4-HWAVGHLM-NH2 - 5.5
TABLE 17
As expected, results from Table 17 show that the universal agonists (L222 &
L223) compete
reasonably well at ~ 50 nM level.
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Name Number Sequence IC50
C6/NMB-
PC3/GRP-
R R
Universal D03A-monoamide-G-Abz4-
a onist L222 y WAV-Ala2-HF-Nle-NH2 - 46
Universal D03A-monoamide-G-Abz4-
a onist L224 F WAV-Ala2-HF-Nle-NH2 - 52
EXAMPLE LXI - NMR Structural Comparison of ~~sLu-L70 and I~sLu-D03A-
monoamide-Aoc-QWAVGHLM-NHZ
[00435} The purpose of this NMR study was to provide complete structural
characterization of Lu-L70 and compare it to the structure of I~SLu-DOTA-Aoc-
QWAVGHLM. L70 and ~~SLu- DOTA-Aoc-QWAVGHLM are both bombesin analogues
(see Figures 60 and 61), differing only in the linker between the chelating
group and the
targeting peptide. In L70 there is a glycyl-4-aminobenzoyl group, whereas in
I~SLu-DOTA-
Aoc-QWAVGHLM there is an S-aminooctanoyl group. However, the biological data
of
these two compounds is strikingly different. Detailed NMR studies were
performed to
explain this difference.
A. EXPERIMENTAL
1. Materials
[00436} 5 mg of I~SLu-D03A-monoamide-Aoc-QWAVGHLM-NH2 was dissolved in 225
uL of DMSO-d6 (Aldrich 100% atom %D).
5 mg of l~sLu-L70 was dissolved in 225 uL of DMSO-d6 (Aldrich 100% atom %D).
2. Acquisition of NMR Data
[00437} All NMR experiments were performed on a Varian Inova-500 Fourier
Transform
NMR spectrometer equipped with a 3 mm broad-band inverse (z-axis gradient)
probe. The
chemical shifts were referenced to the residual CH peaks of DMSO-d6 at 2.50
ppm for the
proton and 40.19 ppm for 13C. The sample temperatures were controlled by a
Varian digital
temperature controller. The data were processed using NMRPipe, VNMR, PROSA,
and
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158
VNMRJ software on the Sun Blade 2000 Unix computer and analyzed using NMRView
and
SPARI~Y software on the Linux computer. The modeling of the peptides was
performed .
employing CYANA software on the Linux computer and further analyzed using
MOLMOL
software on a Compaq Deskpro Workstation.
B. RESULTS and DISCUSSION
[00438 The proton chemical shifts of ~~SLu-L70 were assigned as follows. A
quick survey
of the methyl region (0.5 to 2.5 ppm) in the 1D spectrum allowed the
identification of a sharp
singlet at 2.02 ppm as the methyl peak of methionine. In the same region of
the TOCSY
spectrum, the chemical shift at 1.16 ppm which correlates to only one peak at
4.32 ppm
indicates that they belong to alanine. The methyl peaks at 0.84 and 0.85 ppm
which correlate
to two peaks at 1.98 and 4.12 ppm must belong to valine. The remaining methyl
peaks at
0.84 and 0.88 ppm which correlate to peaks at 1.60, 1.48, and 4.23 ppm belong
to leucine.
These chemical shifts and the chemical shifts of other amino acids are also
present in the
"fingerprint" region (see Wuthrich, K. "NMR of Proteins and Nucleic Acids,"
John Wiley &
Sons, 1986) - the backbone NH-aH region of the TOCSY spectrum (see FIG. 52).
All the
chemical shifts belonging to a spin system of an amino acid will align
themselves vertically.
After a careful examination of the spectrum, all chemical shifts were
assigned. The chemical
shifts were further verified by reviewing other spectra such as COSY (see FIG.
53) and
NOESY (see FIG. 54). After the proton chemical shifts were assigned, their
carbon chemical
shifts were identified through the gHSQC spectrum (see FIG. 55) and further
verified by
reviewing the gHMBC (see FIG. 56) and gHSQCTOCSY (see FIG. 57) spectra. The
chemical shifts of Lu-L70 are listed in Table 19 (the atom numbers are
referenced to FIG.
60).
[00439] Interestingly, in the TOCSY spectrum of l~SLu-L70, the chemical shift
of the NH
proton at 14.15 ppm shows strong correlations to two other peaks of the
histidine ring, and
also to a water molecule. This water molecule is not freely exchanging and is
clearly seen in
the NMR timeframe. To see which proton of the histidine interacts more
strongly with the
water molecule, a selective homo-decoupling experiment was performed on the
~~SLu-L70 at
15 °C. When the water peak was selectively saturated with a low power,
the intensities of the
NH peaks of histidine at 14.16 and 14.23 ppm were dramatically reduced while
the
intensities of the two remaining peaks of histidine at 7.32 and 8.90 ppm were
partially
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reduced (see FIG. 58). The observation of the water protons on the NMR time
scale suggests
a rigid confirmation.
[00440] A proposed chemical structure of ~~$Lu-L70 with a water molecule can
be seen in
FIG. 62. A water molecule occupies a ninth coordination site by capping the
square plane
described by the coordinated oxygens. This has other precedents. Coordination
of water at
the ninth site of Lu in Na[Lu(DOTA)H20)]~4Hz0 was observed in an x-ray
structure, as
shown by Aime et al, Inorg. Chim. Acta 1996, 246, 423-429, which is
incorporated by
reference.
[00441] In contrast, in the TOCSY spectrum of'~SLu-DO3A-monoamide-Aoc-
QWAVGHLM-NH2, the chemical shift of the NH proton only shows strong
correlations to
two other peaks of the histidine ring, but not to the water molecule (see FIG.
59). This
indicates that there is no water molecule simultaneously coordinating both the
I~SLu and the
His-NH in'~SLu-D03A-monoamide-Aoc-QWAVGHLM-NH2. Thus, the difference between
the two molecules is significant. In the I~SLu-L70 a secondary structure of
the peptide is
stabilized via the bound water molecule, and this may be responsible for
increased in vivo
stability.
TABLE 19 - Chemical Shifts (ppm) of ~~SLu-L70 in I?MSO-d6 at 25 °C
Position Chemical Shift
Assi nment Proton Carbon
2/12 -
3/11 -
5/9 -
6/8 -
13 -
15 -
-
17 3.69/3.62
22 9.95/9.73
23 4.04/4.16 (43.57)
26 10.47
28/32 7.62 (118.9)
29/31 7.79 (128.7)
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Position Chemical Shift
Assi nment Proton Carbon
35a 8.54
36 4.29 (54.26)
39 1.83/1.91 (27.26)
40 2.16 (32.08)
47 6.84/7.3 0
43 7.97
44 4.54 (53.37)
48 2.98/3.12 (27.74)
SO 7.12 (123.9)
51 10.79
53 7.53 (118.7)
54 6.93 ( 118.6)
55 7.03 (121.3)
56 7.28 (111.7)
58 8.09
59 4.32 (48.71)
62 1.16 (17.86)
63 7.65
64 4.12 (58.28)
67 1.98 (30.96)
68/73 0.84 (18.42)
0.85 (19.52)
69 8.19
70 3.70/3.74 (42.45)
74 8.10
75 4.60 (51.85)
78 2.95/3.08 (27.50)
80 14.15
81 8.91
83 7.32
84 8.14
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Position Chemical Shift
Assi nment Proton Carbon
85 4.23 (51.93)
86 1.48 (40.6)
87 1.60 (24.61
88/91 0.84 (21.8)
0.88 (23.41)
92 8.04
93 4.25 (52.25)
96 1.76/1.92 (32.16)
97 2.4I (29.91 )
99 2.02 (15.13)
