U.S. patent application number 17/132552 was filed with the patent office on 2021-04-29 for trifunctional constructs with tunable pharmacokinetics useful in imaging and anti-tumor therapies.
This patent application is currently assigned to Cornell University. The applicant listed for this patent is Cornell University. Invention is credited to Alejandro Amor-Coarasa, John W. Babich, James M. Kelly, Shashikanth Ponnala.
Application Number | 20210121584 17/132552 |
Document ID | / |
Family ID | 1000005316164 |
Filed Date | 2021-04-29 |
![](/patent/app/20210121584/US20210121584A1-20210429\US20210121584A1-2021042)
United States Patent
Application |
20210121584 |
Kind Code |
A1 |
Babich; John W. ; et
al. |
April 29, 2021 |
TRIFUNCTIONAL CONSTRUCTS WITH TUNABLE PHARMACOKINETICS USEFUL IN
IMAGING AND ANTI-TUMOR THERAPIES
Abstract
The present technology provides compounds, as well as
compositions including such compounds, useful for imaging and/or
treatment of a glioma, a breast cancer, an adrenal cortical cancer,
a cervical carcinoma, a vulvar carcinoma, an endometrial carcinoma,
a primary ovarian carcinoma, a metastatic ovarian carcinoma, a
non-small cell lung cancer, a small cell lung cancer, a bladder
cancer, a colon cancer, a primary, gastric adenocarcinoma, a
primary colorectal adenocarcinoma, a renal cell carcinoma, and/or a
prostate cancer.
Inventors: |
Babich; John W.; (New York,
NY) ; Kelly; James M.; (New York, NY) ;
Amor-Coarasa; Alejandro; (New York, NY) ; Ponnala;
Shashikanth; (New York, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Cornell University |
Ithaca |
NY |
US |
|
|
Assignee: |
Cornell University
Ithaca
NY
|
Family ID: |
1000005316164 |
Appl. No.: |
17/132552 |
Filed: |
December 23, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17016189 |
Sep 9, 2020 |
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17132552 |
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16134789 |
Sep 18, 2018 |
10806806 |
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17016189 |
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15630808 |
Jun 22, 2017 |
10179117 |
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16134789 |
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PCT/US2018/026340 |
Apr 5, 2018 |
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16134789 |
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62353735 |
Jun 23, 2016 |
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62482038 |
Apr 5, 2017 |
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62574720 |
Oct 19, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 51/0497 20130101;
A61P 35/00 20180101; A61K 51/0402 20130101; A61K 51/0482
20130101 |
International
Class: |
A61K 51/04 20060101
A61K051/04; A61P 35/00 20060101 A61P035/00 |
Goverment Interests
U.S. GOVERNMENT LICENSE RIGHTS
[0002] This invention was made with government support under grant
number UL1TR00457 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A composition comprising: a tumor-binding domain, an
albumin-binding domain, and an imaging agent-containing domain,
wherein the tumor-binding domain comprises an active site that is
distal to and sterically unimpeded by the albumin-binding domain
and the imaging agent-containing domain, and the relative affinity
of the tumor-binding domain and the albumin-binding domain differ
in specific affinity by a factor of at least 100 to about
10,000.
2. The compound of claim 1, wherein the tumor-binding domain binds
to a tumor associated molecular target selected from one or more of
a tumor-specific cell surface protein, prostate specific membrane
antigen (PSMA), somatostatin peptide receptor-2 (SSTR2),
alphavbeta3 (.alpha.v.beta.3), alphavbeta6, a gastrin-releasing
peptide receptor, a seprase, fibroblast activation protein alpha
(FAP-alpha), an incretin receptor, a glucose-dependent
insulinotropic polypeptide receptor, VIP-1, NPY, a folate receptor,
LHRH, a neuronal transporter (e.g., noradrenaline transporter
(NET)), EGFR, HER-2, VGFR, MUC-1, CEA, MUC-4, ED2, TF antigen, an
endothelial specific marker, neuropeptide Y, uPAR, TAG-72, a
claudin, a CCK analog, VIP, bombesin, VEGFR, a tumor-specific cell
surface protein, GLP-1, CXCR4, Hepsin, TMPRSS2, a caspace, cMET, or
an overexpressed peptide receptor.
3. The compound of claim 2, wherein the tumor-binding domain binds
to the tumor associated molecular target with moderate to high
affinity.
4. The compound of claim 1, wherein the imaging agent-containing
domain further comprises an imaging isotope.
5. A compound comprising: a multi-targeted agent having a plurality
of sterically unimpeded targeting domains, comprising a first
targeting domain comprising a blood-protein binding domain having
specific affinity for binding human serum albumin in the range of
about 0.25 to 50 micromolar, and a second targeting domain
comprising a tumor-binding domain having specific affinity for a
tumor associated molecular target in the range of about 0.1 to 75
nanomolar; wherein the relative affinities of the first and second
targeting domains differ in specific affinity by a factor of at
least 100 to about 10,000; and an imaging agent-containing domain
comprising a positron-emitting isotope.
6. The compound of claim 5, wherein the tumor associated molecular
target is selected from one or more of a tumor-specific cell
surface protein, prostate specific membrane antigen (PSMA),
somatostatin peptide receptor-2 (SSTR2), alphavbeta3
(.alpha.v.beta.3), alphavbeta6, a gastrin releasing peptide
receptor, a seprase, fibroblast activation protein alpha
(FAP-alpha), an incretin receptor, a glucose-dependent
insulinotropic polypeptide receptor, VIP-1, NPY, a folate receptor,
LHRH, a neuronal transporter (e.g., noradrenaline transporter
(NET)), EGFR, HER-2, VGFR, MUC-1, CEA, MUC-4, ED2, TF-antigen, an
endothelial specific marker, neuropeptide Y, uPAR, TAG-72, a
claudin, a CCK analog, VIP, bombesin, VEGFR, a tumor-specific cell
surface protein, GLP-1, CXCR4, Hepsin, TMPRSS2, a caspace, cMET, or
an overexpressed peptide receptor.
7. The compound of claim 6, wherein the tumor-binding domain binds
to the tumor associated molecular target with moderate to high
affinity.
8. The compound of claim 5, wherein the positron-emitting isotope
is .sup.18F or .sup.68Ga.
9. A compound comprising: a multi-targeted agent having a plurality
of sterically unimpeded targeting domains comprising, a first
targeting domain comprising a blood-protein binding domain having
specific affinity for binding human serum albumin in the range of
about 0.25 to 50 micromolar, a second targeting domain comprising a
tumor-binding domain having specific affinity for fibroblast
activation protein alpha (FAP-alpha) in the range of about 0.1 to
75 nanomolar; wherein the relative affinities of the first and
second targeting domains differ in specific affinity by a factor of
at least 100 to about 10,000; and an imaging agent-containing
domain.
10. The compound of claim 9, wherein the blood-protein binding
domain binds human serum albumin with an affinity in the range of
about 0.4 to 20 micromolar, and the tumor-binding domain binds
FAP-alpha with an affinity in the range of about 0.1 to 15
nanomolar; wherein the relative affinities of the first and second
targeting domains differ in specific affinity by a factor of about
1,000 to about 10,000.
11. The compound of claim 9, wherein the blood-protein binding
domain is selected from one or more of myristic acid, a substituted
or unsubstituted indole-2-carboxylic acid, a substituted or
unsubstituted thioamide, a substituted or unsubstituted
4-oxo-4-(5,6,7,8-tetrahydronaphthalen-2-yl)butanoic acid, a
substituted or unsubstituted naphthalene acylsulfonamide, a
substituted or unsubstituted diphenylcyclohexanol phosphate ester,
a substituted or unsubstituted 4-iodophenylalkanoic acid, a
substituted or unsubstituted 3-(4-iodophenyl)propionic acid, a
substituted or unsubstituted 2-(4-iodophenyl)acetic acid, or a
substituted or unsubstituted 4-(4-iodophenyl)butanoic acid.
12. The compound of claim 9, wherein the tumor-binding domain binds
to the FAP-alpha with moderate to high affinity.
13. The compound of claim 9, wherein the imaging agent-containing
domain further comprises a positron-emitting isotope.
14. The compound of claim 9, wherein the positron-emitting isotope
is .sup.18F or .sup.68Ga.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of U.S. patent
application Ser. No. 17/016,189, filed Sep. 9, 2020, which is a
Divisional of U.S. patent application Ser. No. 16/134,789, filed
Sep. 18, 2018, which is a Continuation-in-Part of U.S. patent
application Ser. No. 15/630,808, filed Jun. 22, 2017, now U.S. Pat.
No. 10,179,117, which claims the benefit of priority from U.S.
Provisional Patent Application No. 62/353,735, filed on Jun. 23,
2016. U.S. patent application Ser. No. 16/134,789 is also a
Continuation-in-Part of International Patent Application No.
PCT/US2018/026340, filed Apr. 5, 2018, which claims the benefit of
priority from U.S. Provisional Patent Application No. 62/482,038,
filed Apr. 5, 2017, and U.S. Provisional Patent Application No.
62/574,720, filed Oct. 19, 2017, the entire disclosures of each of
which are incorporated herein by reference for any and all purposes
in their entirety.
FIELD
[0003] The present technology relates to targeted drug delivery,
more particularly compounds useful in anti-tumor therapy. For
example, the compositions described herein may be used as
radiotherapy agents for the treatment of cancers such as prostate
cancer.
SUMMARY
[0004] The present invention details compounds that are useful
agents for the radiotherapy of tumors. The compounds have affinity
for cellular markers associated with neoplastic tissues as well as
blood proteins, thereby providing increased residence time in the
circulatory system of a recipient, which increases tumor perfusion
and achieves greater uptake and compound loading of the tumor, as
well as minimizing compound binding to non-target tissues with
consequent side effects. In one aspect of the present technology, a
multi-targeted compound is provided that includes a tumor-binding
domain, an albumin-binding domain, and a cytocidal or cytostatic
therapeutic agent, where the tumor-binding domain includes an
active site that is distal to and sterically unimpeded by the
albumin-binding domain and the therapeutic agent, and the relative
affinity of the tumor-binding domain and the albumin-binding domain
differ in specific affinity by a factor of at least 100 to about
10,000.
[0005] In a related aspect, a multi-targeted compound is provided,
having a plurality of sterically unimpeded targeting domains, where
the compound includes a first targeting domain that includes a
blood-protein binding domain having specific affinity for binding
human serum albumin in the range of about 0.25 to 50 micromolar, a
second targeting domain that includes a tumor-binding domain having
specific affinity for a tumor associated molecular target in the
range of about 0.1 to 75 nanomolar; wherein the relative affinities
of the first and second targeting domains differ in specific
affinity by a factor of at least 100 to about 10,000; and a
therapeutic domain that includes a cytocidal or cytostatic
therapeutic agent. In a more specific embodiment of the above, the
invention provides for a compound including a multi-targeted agent
having a plurality of sterically unimpeded targeting domains
comprising, a first targeting domain comprising a blood-protein
binding domain having specific affinity for binding human serum
albumin in the range of about 0.25 to 50 micromolar, a second
targeting domain comprising a tumor-binding domain having specific
affinity for PSMA in the range of about 0.1 to 75 nanomolar;
wherein the relative affinities of the first and second targeting
domains differ in specific affinity by a factor of at least 100 to
about 10,000; and a therapeutic domain comprising a cytocidal or
cytostatic therapeutic agent. In various embodiments, the tumor
associated molecular target is selected from one or more of a
tumor-specific cell surface protein, prostate specific membrane
antigen (PSMA), somatostatin peptide receptor-2 (SSTR2),
alphavbeta3 (.alpha.v.beta.3), alphavbeta6, a gastrin-releasing
peptide receptor, a seprase, fibroblast activation protein alpha
(FAP-alpha), an incretin receptor, a glucose-dependent
insulinotropic polypeptide receptor, VIP-1, NPY, a folate receptor,
LHRH, a neuronal transporter (e.g., noradrenaline transporter
(NET)), EGFR, HER-2, VGFR, MUC-1, CEA, MUC-4, ED2, TF-antigen, an
endothelial specific marker, neuropeptide Y, uPAR, TAG-72, a
claudin, a CCK analog, VIP, bombesin, VEGFR, a tumor-specific cell
surface protein, GLP-1, CXCR4, Hepsin, TMPRSS2, a caspace, cMET, or
an overexpressed peptide receptor. The compounds include a
tumor-binding domain that binds to the tumor associated molecular
target with moderate to high affinity (e.g. an affinity constant
(K.sub.D) of approximately 10{circumflex over ( )}-8M to
10{circumflex over ( )}-10M). In various embodiments, the compounds
include a cytocidal or cytostatic therapeutic agent that is a
toxin, a venom, a metabolic poison, a chemotherapeutic agent, an
auger electron-emitting radionuclide, a beta-emitting radionuclide,
or an alpha-emitting radionuclide. In other such embodiments, the
therapeutic domain comprises a covalently conjugated chelating
agent or a covalently conjugated polyaza polycarboxylic macrocycle.
In still other embodiments, the therapeutic domain further
comprises an auger electron-emitting radionuclide, a beta-emitting
radionuclide, or an alpha-emitting radionuclide.
BRIEF DESCRIPTION OF THE DRAWINGS
[0006] FIG. 1 provides PET images of mice injected with
.sup.68Ga-RPS-055 (top) or .sup.68Ga-DKFZ-617 (bottom) both 1 h and
3 h post injection. All images are of the same mouse bearing a
small LNCaP tumor in the right shoulder (arrow). Images were taken
on sequential days using a Siemens Inveon .mu.PET/.mu.CT system
(Siemens Corp., Munich, Germany). All images were windowed to a
maximum of 4% injected dose per cc tissue and are decay corrected
and dose corrected.
[0007] FIG. 2 provides PET images of mice injected with
.sup.68Ga-RPS-055 (left panels) or .sup.68Ga-RPS-056 (right panels)
both 1 h (top) and 3 h (bottom) post injection. All images are of
the same mice bearing small LNCaP tumors in their right shoulders.
Images were taken on sequential days using a Siemens Inveon
.mu.PET/.mu.CT system (Siemens Corp., Munich, Germany). All images
were windowed to a maximum of 20% injected dose per cc tissue and
are decay corrected and dose corrected.
[0008] FIG. 3 provides SPECT images of mice injected with
.sup.177Lu-RPS-055 both 4 h (top) and 3 h (bottom) post injection.
All images are of the same mice bearing small LNCaP tumors in their
right shoulders (left panels and right panels). Images were taken
on the same day using a Siemens Inveon .mu.SPECT/.mu.CT system
(Siemens Corp., Munich, Germany).
[0009] FIG. 4 shows PET images of BALB/C nu/nu mice bearing LNCaP
tumor xenografts and injected intravenously with either
.sup.68Ga-RPS-061, .sup.68Ga-PSMA-617 or .sup.68Ga-RPS-030. The
mice were imaged on sequential days at 1 h (left) and 3 h (right)
post injection using a Siemens Inveon .mu.SPECT/.mu.CT system
(Siemens Corp., Munich, Germany).
[0010] FIGS. 5A-C provide histograms illustrating the
biodistribution of .sup.225Ac(NO.sub.3).sub.3 (FIG. 5A),
[.sup.225Ac(macropa)].sup.+ (FIG. 5B), and [.sup.225Ac(DOTA)].sup.-
(FIG. 5C) for select organs following intravenous injection in
mice. Adult C57BL/6 mice were sacrificed 15 min, 1 h, or 5 h post
injection. Values for each time point are given as mean % ID/g.+-.1
SD.
[0011] FIG. 6 illustrates the biodistribution of
.sup.225Ac-macropa-RPS-070 following intravenous injection in LNCaP
tumor xenograft mice. Mice were sacrificed 4, 24, or 96 h post
injection. Values for each time point are given as mean % ID/g.+-.1
SEM.
[0012] FIG. 7 provides PET images of LNCaP xenograft mice with
.sup.66Ga-labeled tracers at 1 h, 3 h, 6 h and 24 h post injection.
Mice were injected intravenously with a bolus injection of 0.56-5.4
MBq (15-145 .mu.Ci) of the tracer. The total mass of ligand
injected was 4 .mu.g. Prior to imaging, the mice were anesthetized
with isoflurane and then imaged for 30 min. The images were
corrected for decay and for activity injected.
[0013] FIG. 8 provides the biodistribution of .sup.177Lu-RPS-068,
.sup.177Lu-RPS-063, .sup.177Lu-RPS-061, .sup.177Lu-RPS-069,
.sup.177Lu-RPS-066, .sup.177Lu-RPS-067 and .sup.177Lu-PSMA-617.
Male athymic nude mice bearing LNCaP xenograft tumors (n=5 per time
point) were injected intravenously with 348-851 kBq (9.4-23 .mu.Ci)
of the labeled compound and sacrificed at 4 h, 24 h and 96 h p.i.
The total mass of ligand injected was 37-50 ng (23-25 pmol).
[0014] FIG. 9 provides a comparison of the blood pool activity of
different .sup.177Lu-labeled ligands at 4 h, 24 h and 96 h post
injection. Errors are expressed as SEM. RPS ligands are displayed
in order of increasing size.
[0015] FIG. 10 provides time-activity curves (TAC) of tumor and
kidney uptake of .sup.177Lu-PSMA-617, .sup.177Lu-RPS-061,
.sup.177Lu-RPS-063, .sup.177Lu-RPS-066, .sup.177Lu-RPS-067,
.sup.177Lu-RPS-068 and .sup.177Lu-RPS-069 in male athymic nude mice
bearing LNCaP xenograft tumors. Uptake is expressed as % ID/g.
[0016] FIG. 11 provides a comparison of relative dose integral in
the tumor of male LNCaP xenograft tumor-bearing mice injected with
the corresponding .sup.177Lu-labeled compounds and studied over 96
h. Values are normalized to .sup.177Lu-PSMA-617.
[0017] FIG. 12 shows the uptake of activity in blood, normal tissue
and tumor in male BALB/C nu/nu mice bearing LNCaP xenograft tumors.
Mice (n=4/time point) were injected intravenously with 105 kBq
.sup.225Ac-RPS-074 and sacrificed at 4 h, 24 h, 7 d, 14 d and 21 d
p.i.
[0018] FIG. 13 plots the change in average tumor volumes of
individual male BALB/C nu/nu mice bearing LNCaP xenograft tumors
and treated with a) 138 kBq .sup.225Ac-RPS-074; b) 74 kBq
.sup.225Ac-RPS-074; c) 37 kBq .sup.225Ac-RPS-074; d) 133 kBq
.sup.225Ac-DOTA-Lys-IPBA; and e) vehicle.
[0019] FIG. 14 provides .sup.68Ga-PSMA-11 .mu.PET/CT images of mice
treated with 138 kBq .sup.225Ac-RPS-074 (left) or 74 kBq
.sup.225Ac-RPS-074 (right). Images were acquired 60 min post
injection and are corrected for decay and for activity
injected.
[0020] FIG. 15 plots a Kaplan-Meier curve illustrating the overall
survival of the mice. ##=Activity of .sup.225Ac-DOTA-Lys-IPBA
administered. Mice were sacrificed when tumor volume exceeded 2000
mm.sup.3.
DETAILED DESCRIPTION
[0021] The following terms are used throughout as defined
below.
[0022] As used herein and in the appended claims, singular articles
such as "a" and "an" and "the" and similar referents in the context
of describing the elements (especially in the context of the
following claims) are to be construed to cover both the singular
and the plural, unless otherwise indicated herein or clearly
contradicted by context. Recitation of ranges of values herein are
merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range,
unless otherwise indicated herein, and each separate value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the embodiments and does not
pose a limitation on the scope of the claims unless otherwise
stated. No language in the specification should be construed as
indicating any non-claimed element as essential.
[0023] As used herein, "about" will be understood by persons of
ordinary skill in the art and will vary to some extent depending
upon the context in which it is used. If there are uses of the term
which are not clear to persons of ordinary skill in the art, given
the context in which it is used, "about" will mean up to plus or
minus 10% of the particular term.
[0024] Generally, reference to a certain element such as hydrogen
or H is meant to include all isotopes of that element. For example,
if an R group is defined to include hydrogen or H, it also includes
deuterium and tritium. Compounds comprising radioisotopes such as
tritium, C.sup.14, P.sup.32 and S.sup.35 are thus within the scope
of the present technology. Procedures for inserting such labels
into the compounds of the present technology will be readily
apparent to those skilled in the art based on the disclosure
herein.
[0025] In general, "substituted" refers to an organic group as
defined below (e.g., an alkyl group) in which one or more bonds to
a hydrogen atom contained therein are replaced by a bond to
non-hydrogen or non-carbon atoms. Substituted groups also include
groups in which one or more bonds to a carbon(s) or hydrogen(s)
atom are replaced by one or more bonds, including double or triple
bonds, to a heteroatom. Thus, a substituted group is substituted
with one or more substituents, unless otherwise specified. In some
embodiments, a substituted group is substituted with 1, 2, 3, 4, 5,
or 6 substituents. Examples of substituent groups include: halogens
(i.e., F, Cl, Br, and I); hydroxyls; alkoxy, alkenoxy, aryloxy,
aralkyloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, and
heterocyclylalkoxy groups; carbonyls (oxo); carboxylates; esters;
urethanes; oximes; hydroxylamines; alkoxyamines; aralkoxyamines;
thiols; sulfides; sulfoxides; sulfones; sulfonyls;
pentafluorosulfanyl (i.e., SF.sub.5), sulfonamides; amines;
N-oxides; hydrazines; hydrazides; hydrazones; azides; amides;
ureas; amidines; guanidines; enamines; imides; isocyanates;
isothiocyanates; cyanates; thiocyanates; imines; nitro groups;
nitriles (i.e., CN); and the like.
[0026] Substituted ring groups such as substituted cycloalkyl,
aryl, heterocyclyl and heteroaryl groups also include rings and
ring systems in which a bond to a hydrogen atom is replaced with a
bond to a carbon atom. Therefore, substituted cycloalkyl, aryl,
heterocyclyl and heteroaryl groups may also be substituted with
substituted or unsubstituted alkyl, alkenyl, and alkynyl groups as
defined below.
[0027] As used herein, C.sub.m-C.sub.n, such as C.sub.1-C.sub.12,
C.sub.1-C.sub.8, or C.sub.1-C.sub.6 when used before a group refers
to that group containing m to n carbon atoms.
[0028] Alkyl groups include straight chain and branched chain alkyl
groups having from 1 to 12 carbon atoms, and typically from 1 to 10
carbons or, in some embodiments, from 1 to 8, 1 to 6, or 1 to 4
carbon atoms. Examples of straight chain alkyl groups include
groups such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl,
n-heptyl, and n-octyl groups. Examples of branched alkyl groups
include, but are not limited to, isopropyl, iso-butyl, sec-butyl,
tert-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups.
Alkyl groups may be substituted or unsubstituted. Representative
substituted alkyl groups may be substituted one or more times with
substituents such as those listed above, and include without
limitation haloalkyl (e.g., trifluoromethyl), hydroxyalkyl,
thioalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl,
alkoxyalkyl, carboxyalkyl, and the like.
[0029] Cycloalkyl groups include mono-, bi- or tricyclic alkyl
groups having from 3 to 12 carbon atoms in the ring(s), or, in some
embodiments, 3 to 10, 3 to 8, or 3 to 4, 5, or 6 carbon atoms.
Exemplary monocyclic cycloalkyl groups include, but not limited to,
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and
cyclooctyl groups. In some embodiments, the cycloalkyl group has 3
to 8 ring members, whereas in other embodiments the number of ring
carbon atoms range from 3 to 5, 3 to 6, or 3 to 7. Bi- and
tricyclic ring systems include both bridged cycloalkyl groups and
fused rings, such as, but not limited to, bicyclo[2.1.1]hexane,
adamantyl, decalinyl, and the like. Cycloalkyl groups may be
substituted or unsubstituted. Substituted cycloalkyl groups may be
substituted one or more times with, non-hydrogen and non-carbon
groups as defined above. However, substituted cycloalkyl groups
also include rings that are substituted with straight or branched
chain alkyl groups as defined above. Representative substituted
cycloalkyl groups may be mono-substituted or substituted more than
once, such as, but not limited to, 2,2-, 2,3-, 2,4- 2,5- or
2,6-disubstituted cyclohexyl groups, which may be substituted with
substituents such as those listed above.
[0030] Cycloalkylalkyl groups are alkyl groups as defined above in
which a hydrogen or carbon bond of an alkyl group is replaced with
a bond to a cycloalkyl group as defined above. In some embodiments,
cycloalkylalkyl groups have from 4 to 16 carbon atoms, 4 to 12
carbon atoms, and typically 4 to 10 carbon atoms. Cycloalkylalkyl
groups may be substituted or unsubstituted. Substituted
cycloalkylalkyl groups may be substituted at the alkyl, the
cycloalkyl or both the alkyl and cycloalkyl portions of the group.
Representative substituted cycloalkylalkyl groups may be
mono-substituted or substituted more than once, such as, but not
limited to, mono-, di- or tri-substituted with substituents such as
those listed above.
[0031] Alkenyl groups include straight and branched chain alkyl
groups as defined above, except that at least one double bond
exists between two carbon atoms. Alkenyl groups have from 2 to 12
carbon atoms, and typically from 2 to 10 carbons or, in some
embodiments, from 2 to 8, 2 to 6, or 2 to 4 carbon atoms. In some
embodiments, the alkenyl group has one, two, or three carbon-carbon
double bonds. Examples include, but are not limited to vinyl,
allyl, --CH.dbd.CH(CH.sub.3), --CH.dbd.C(CH.sub.3).sub.2,
--C(CH.sub.3).dbd.CH.sub.2, --C(CH.sub.3).dbd.CH(CH.sub.3),
--C(CH.sub.2CH.sub.3).dbd.CH.sub.2, among others. Alkenyl groups
may be substituted or unsubstituted. Representative substituted
alkenyl groups may be mono-substituted or substituted more than
once, such as, but not limited to, mono-, di- or tri-substituted
with substituents such as those listed above.
[0032] Cycloalkenyl groups include cycloalkyl groups as defined
above, having at least one double bond between two carbon atoms.
Cycloalkenyl groups may be substituted or unsubstituted. In some
embodiments the cycloalkenyl group may have one, two or three
double bonds but does not include aromatic compounds. Cycloalkenyl
groups have from 4 to 14 carbon atoms, or, in some embodiments, 5
to 14 carbon atoms, 5 to 10 carbon atoms, or even 5, 6, 7, or 8
carbon atoms. Examples of cycloalkenyl groups include cyclohexenyl,
cyclopentenyl, cyclohexadienyl, cyclobutadienyl, and
cyclopentadienyl.
[0033] Cycloalkenylalkyl groups are alkyl groups as defined above
in which a hydrogen or carbon bond of the alkyl group is replaced
with a bond to a cycloalkenyl group as defined above.
Cycloalkenylalkyl groups may be substituted or unsubstituted.
Substituted cycloalkenylalkyl groups may be substituted at the
alkyl, the cycloalkenyl or both the alkyl and cycloalkenyl portions
of the group. Representative substituted cycloalkenylalkyl groups
may be substituted one or more times with substituents such as
those listed above.
[0034] Alkynyl groups include straight and branched chain alkyl
groups as defined above, except that at least one triple bond
exists between two carbon atoms. Alkynyl groups have from 2 to 12
carbon atoms, and typically from 2 to 10 carbons or, in some
embodiments, from 2 to 8, 2 to 6, or 2 to 4 carbon atoms. In some
embodiments, the alkynyl group has one, two, or three carbon-carbon
triple bonds. Examples include, but are not limited to
--C.ident.CH, --C.ident.CCH.sub.3, --CH.sub.2C.ident.CCH.sub.3,
--C.ident.CCH.sub.2CH(CH.sub.2CH.sub.3).sub.2, among others.
Alkynyl groups may be substituted or unsubstituted. Representative
substituted alkynyl groups may be mono-substituted or substituted
more than once, such as, but not limited to, mono-, di- or
tri-substituted with substituents such as those listed above.
[0035] Aryl groups are cyclic aromatic hydrocarbons that do not
contain heteroatoms. Aryl groups herein include monocyclic,
bicyclic and tricyclic ring systems. Thus, aryl groups include, but
are not limited to, phenyl, azulenyl, heptalenyl, biphenyl,
fluorenyl, phenanthrenyl, anthracenyl, indenyl, indanyl,
pentalenyl, and naphthyl groups. In some embodiments, aryl groups
contain 6-14 carbons, and in others from 6 to 12 or even 6-10
carbon atoms in the ring portions of the groups. In some
embodiments, the aryl groups are phenyl or naphthyl. Aryl groups
may be substituted or unsubstituted. The phrase "aryl groups"
includes groups containing fused rings, such as fused
aromatic-aliphatic ring systems (e.g., indanyl, tetrahydronaphthyl,
and the like). Representative substituted aryl groups may be
mono-substituted or substituted more than once. For example,
monosubstituted aryl groups include, but are not limited to, 2-,
3-, 4-, 5-, or 6-substituted phenyl or naphthyl groups, which may
be substituted with substituents such as those listed above.
[0036] Aralkyl groups are alkyl groups as defined above in which a
hydrogen or carbon bond of an alkyl group is replaced with a bond
to an aryl group as defined above. In some embodiments, aralkyl
groups contain 7 to 16 carbon atoms, 7 to 14 carbon atoms, or 7 to
10 carbon atoms. Aralkyl groups may be substituted or
unsubstituted. Substituted aralkyl groups may be substituted at the
alkyl, the aryl or both the alkyl and aryl portions of the group.
Representative aralkyl groups include but are not limited to benzyl
and phenethyl groups and fused (cycloalkylaryl)alkyl groups such as
4-indanylethyl. Representative substituted aralkyl groups may be
substituted one or more times with substituents such as those
listed above.
[0037] Heterocyclyl groups include aromatic (also referred to as
heteroaryl) and non-aromatic ring compounds containing 3 or more
ring members, of which one or more is a heteroatom such as, but not
limited to, N, O, and S. In some embodiments, the heterocyclyl
group contains 1, 2, 3 or 4 heteroatoms. In some embodiments,
heterocyclyl groups include mono-, bi- and tricyclic rings having 3
to 16 ring members, whereas other such groups have 3 to 6, 3 to 10,
3 to 12, or 3 to 14 ring members. Heterocyclyl groups encompass
aromatic, partially unsaturated and saturated ring systems, such
as, for example, imidazolyl, imidazolinyl and imidazolidinyl
groups. The phrase "heterocyclyl group" includes fused ring species
including those comprising fused aromatic and non-aromatic groups,
such as, for example, benzotriazolyl,
2,3-dihydrobenzo[1,4]dioxinyl, and benzo[1,3]dioxolyl. The phrase
also includes bridged polycyclic ring systems containing a
heteroatom such as, but not limited to, quinuclidyl. Heterocyclyl
groups may be substituted or unsubstituted. Heterocyclyl groups
include, but are not limited to, aziridinyl, azetidinyl,
pyrrolidinyl, imidazolidinyl, pyrazolidinyl, thiazolidinyl,
tetrahydrothiophenyl, tetrahydrofuranyl, dioxolyl, furanyl,
thiophenyl, pyrrolyl, pyrrolinyl, imidazolyl, imidazolinyl,
pyrazolyl, pyrazolinyl, triazolyl, tetrazolyl, oxazolyl,
isoxazolyl, thiazolyl, thiazolinyl, isothiazolyl, thiadiazolyl,
oxadiazolyl, piperidyl, piperazinyl, morpholinyl, thiomorpholinyl,
tetrahydropyranyl, tetrahydrothiopyranyl, oxathiane, dioxyl,
dithianyl, pyranyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl,
triazinyl, dihydropyridyl, dihydrodithiinyl, dihydrodithionyl,
homopiperazinyl, quinuclidyl, indolyl, indolinyl, isoindolyl,
azaindolyl (pyrrolopyridyl), indazolyl, indolizinyl,
benzotriazolyl, benzimidazolyl, benzofuranyl, benzothiophenyl,
benzthiazolyl, benzoxadiazolyl, benzoxazinyl, benzodithiinyl,
benzoxathiinyl, benzothiazinyl, benzoxazolyl, benzothiazolyl,
benzothiadiazolyl, benzo[1,3]dioxolyl, pyrazolopyridyl,
imidazopyridyl (azabenzimidazolyl), triazolopyridyl,
isoxazolopyridyl, purinyl, xanthinyl, adeninyl, guaninyl,
quinolinyl, isoquinolinyl, quinolizinyl, quinoxalinyl,
quinazolinyl, cinnolinyl, phthalazinyl, naphthyridinyl, pteridinyl,
thianaphthyl, dihydrobenzothiazinyl, dihydrobenzofuranyl,
dihydroindolyl, dihydrobenzodioxinyl, tetrahydroindolyl,
tetrahydroindazolyl, tetrahydrobenzimidazolyl,
tetrahydrobenzotriazolyl, tetrahydropyrrolopyridyl,
tetrahydropyrazolopyridyl, tetrahydroimidazopyridyl,
tetrahydrotriazolopyridyl, and tetrahydroquinolinyl groups.
Representative substituted heterocyclyl groups may be
mono-substituted or substituted more than once, such as, but not
limited to, pyridyl or morpholinyl groups, which are 2-, 3-, 4-,
5-, or 6-substituted, or disubstituted with various substituents
such as those listed above.
[0038] Heteroaryl groups are aromatic ring compounds containing 5
or more ring members, of which, one or more is a heteroatom such
as, but not limited to, N, O, and S. Heteroaryl groups include, but
are not limited to, groups such as pyrrolyl, pyrazolyl, triazolyl,
tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl,
pyridazinyl, pyrimidinyl, pyrazinyl, thiophenyl, benzothiophenyl,
furanyl, benzofuranyl, indolyl, azaindolyl (pyrrolopyridinyl),
indazolyl, benzimidazolyl, imidazopyridinyl (azabenzimidazolyl),
pyrazolopyridinyl, triazolopyridinyl, benzotriazolyl, benzoxazolyl,
benzothiazolyl, benzothiadiazolyl, imidazopyridinyl,
isoxazolopyridinyl, thianaphthyl, purinyl, xanthinyl, adeninyl,
guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl,
quinoxalinyl, and quinazolinyl groups. Heteroaryl groups include
fused ring compounds in which all rings are aromatic such as
indolyl groups and include fused ring compounds in which only one
of the rings is aromatic, such as 2,3-dihydro indolyl groups.
Heteroaryl groups may be substituted or unsubstituted. Thus, the
phrase "heteroaryl groups" includes fused ring compounds as well as
includes heteroaryl groups that have other groups bonded to one of
the ring members, such as alkyl groups. Representative substituted
heteroaryl groups may be substituted one or more times with various
substituents such as those listed above.
[0039] Heterocyclylalkyl groups are alkyl groups as defined above
in which a hydrogen or carbon bond of an alkyl group is replaced
with a bond to a heterocyclyl group as defined above.
Heterocyclylalkyl groups may be substituted or unsubstituted.
Substituted heterocyclylalkyl groups may be substituted at the
alkyl, the heterocyclyl or both the alkyl and heterocyclyl portions
of the group. Representative heterocyclyl alkyl groups include, but
are not limited to, morpholin-4-yl-ethyl, furan-2-yl-methyl,
imidazol-4-yl-methyl, pyridin-3-yl-methyl,
tetrahydrofuran-2-yl-ethyl, and indol-2-yl-propyl. Representative
substituted heterocyclylalkyl groups may be substituted one or more
times with substituents such as those listed above.
[0040] Heteroaralkyl groups are alkyl groups as defined above in
which a hydrogen or carbon bond of an alkyl group is replaced with
a bond to a heteroaryl group as defined above. Heteroaralkyl groups
may be substituted or unsubstituted. Substituted heteroaralkyl
groups may be substituted at the alkyl, the heteroaryl or both the
alkyl and heteroaryl portions of the group. Representative
substituted heteroaralkyl groups may be substituted one or more
times with substituents such as those listed above.
[0041] Groups described herein having two or more points of
attachment (i.e., divalent, trivalent, or polyvalent) within the
compound of the present technology are designated by use of the
suffix, "ene." For example, divalent alkyl groups are alkylene
groups, divalent aryl groups are arylene groups, divalent
heteroaryl groups are divalent heteroarylene groups, and so forth.
Substituted groups having a single point of attachment to the
compound of the present technology are not referred to using the
"ene" designation. Thus, e.g., chloroethyl is not referred to
herein as chloroethylene. Such groups may further be substituted or
unsubstituted.
[0042] Alkoxy groups are hydroxyl groups (--OH) in which the bond
to the hydrogen atom is replaced by a bond to a carbon atom of a
substituted or unsubstituted alkyl group as defined above. Examples
of linear alkoxy groups include but are not limited to methoxy,
ethoxy, propoxy, butoxy, pentoxy, hexoxy, and the like. Examples of
branched alkoxy groups include but are not limited to isopropoxy,
sec-butoxy, tert-butoxy, isopentoxy, isohexoxy, and the like.
Examples of cycloalkoxy groups include but are not limited to
cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and
the like. Alkoxy groups may be substituted or unsubstituted.
Representative substituted alkoxy groups may be substituted one or
more times with substituents such as those listed above.
[0043] The terms "alkanoyl" and "alkanoyloxy" as used herein can
refer, respectively, to --C(O)-alkyl and --O--C(O)-alkyl groups,
where in some embodiments the alkanoyl or alkanoyloxy groups each
contain 2-5 carbon atoms. Similarly, the terms "aryloyl" and
"aryloyloxy" respectively refer to --C(O)-aryl and --O--C(O)-aryl
groups.
[0044] The terms "aryloxy" and "arylalkoxy" refer to, respectively,
a substituted or unsubstituted aryl group bonded to an oxygen atom
and a substituted or unsubstituted aralkyl group bonded to the
oxygen atom at the alkyl. Examples include but are not limited to
phenoxy, naphthyloxy, and benzyloxy. Representative substituted
aryloxy and arylalkoxy groups may be substituted one or more times
with substituents such as those listed above.
[0045] The term "carboxylic acid" as used herein refers to a
compound with a --C(O)OH group. The term "carboxylate" as used
herein refers to a --C(O)O-- group. A "protected carboxylate"
refers to a --C(O)O-G where G is a carboxylate protecting group.
Carboxylate protecting groups are well known to one of ordinary
skill in the art. An extensive list of protecting groups for the
carboxylate group functionality may be found in Protective Groups
in Organic Synthesis, Greene, T. W.; Wuts, P. G. M., John Wiley
& Sons, New York, N.Y., (3rd Edition, 1999) which can be added
or removed using the procedures set forth therein and which is
hereby incorporated by reference in its entirety and for any and
all purposes as if fully set forth herein.
[0046] The term "ester" as used herein refers to --COOR.sup.70
groups. R.sup.70 is a substituted or unsubstituted alkyl,
cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heterocyclylalkyl or
heterocyclyl group as defined herein.
[0047] The term "amide" (or "amido") includes C- and N-amide
groups, i.e., --C(O)NR.sup.71R.sup.72, and --NR.sup.71C(O)R.sup.72
groups, respectively. R.sup.71 and R.sup.72 are independently
hydrogen, or a substituted or unsubstituted alkyl, alkenyl,
alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl or
heterocyclyl group as defined herein. Amido groups therefore
include but are not limited to carbamoyl groups (--C(O)NH.sub.2)
and formamide groups (--NHC(O)H). In some embodiments, the amide is
--NR.sup.71C(O)--(C.sub.1-5 alkyl) and the group is termed
"carbonylamino," and in others the amide is --NHC(O)-alkyl and the
group is termed "alkanoylamino."
[0048] The term "nitrile" or "cyano" as used herein refers to the
--CN group.
[0049] Urethane groups include N- and O-urethane groups, i.e.,
--NR.sup.73C(O)OR.sup.74 and --OC(O)NR.sup.73R.sup.74 groups,
respectively. R.sup.73 and R.sup.74 are independently a substituted
or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl,
aralkyl, heterocyclylalkyl, or heterocyclyl group as defined
herein. R.sup.73 may also be H.
[0050] The term "amine" (or "amino") as used herein refers to
--NR.sup.75R.sup.76 groups, wherein R.sup.75 and R.sup.76 are
independently hydrogen, or a substituted or unsubstituted alkyl,
alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl or
heterocyclyl group as defined herein. In some embodiments, the
amine is alkylamino, dialkylamino, arylamino, or alkylarylamino. In
other embodiments, the amine is NH.sub.2, methylamino,
dimethylamino, ethylamino, diethylamino, propylamino,
isopropylamino, phenylamino, or benzylamino.
[0051] The term "sulfonamido" includes S- and N-sulfonamide groups,
i.e., --SO.sub.2NR.sup.78R.sup.79 and --NR.sup.78SO.sub.2R.sup.79
groups, respectively. R.sup.78 and R.sup.79 are independently
hydrogen, or a substituted or unsubstituted alkyl, alkenyl,
alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl, or
heterocyclyl group as defined herein. Sulfonamido groups therefore
include but are not limited to sulfamoyl groups
(--SO.sub.2NH.sub.2). In some embodiments herein, the sulfonamido
is --NHSO.sub.2-alkyl and is referred to as the
"alkylsulfonylamino" group.
[0052] The term "thiol" refers to --SH groups, while sulfides
include --SR.sup.8 groups, sulfoxides include --S(O)R.sup.81
groups, sulfones include --SO.sub.2R.sup.82 groups, and sulfonyls
include --SO.sub.20R.sup.83. R.sup.80, R.sup.81, R.sup.82, and
R.sup.83 are each independently a substituted or unsubstituted
alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or
heterocyclylalkyl group as defined herein. In some embodiments the
sulfide is an alkylthio group, --S-alkyl.
[0053] The term "urea" refers to
--NR.sup.84--C(O)--NR.sup.85R.sup.86 groups. R.sup.84, R.sup.85,
and R.sup.86 groups are independently hydrogen, or a substituted or
unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl,
heterocyclyl, or heterocyclylalkyl group as defined herein.
[0054] The term "amidine" refers to --C(NR.sup.87)NR.sup.88R.sup.89
and --NR.sup.87C(NR.sup.88)R.sup.89, wherein R.sup.87, R.sup.88,
and R.sup.89 are each independently hydrogen, or a substituted or
unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl,
heterocyclyl or heterocyclylalkyl group as defined herein.
[0055] The term "guanidine" refers to
--NR.sup.90C(NR.sup.91)NR.sup.92R.sup.93, wherein R.sup.90,
R.sup.91, R.sup.92 and R.sup.93 are each independently hydrogen, or
a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl,
aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined
herein.
[0056] The term "enamine" refers to
--C(R.sup.94).dbd.C(R.sup.95)NR.sup.96R.sup.97 and
--NR.sup.94C(R.sup.95).dbd.C(R.sup.96)R.sup.97, wherein R.sup.94,
R.sup.95, R.sup.96 and R.sup.97 are each independently hydrogen, a
substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl,
aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined
herein.
[0057] The term "halogen" or "halo" as used herein refers to
bromine, chlorine, fluorine, or iodine. In some embodiments, the
halogen is fluorine. In other embodiments, the halogen is chlorine
or bromine.
[0058] The term "hydroxyl" as used herein can refer to --OH or its
ionized form, --O.sup.-.
[0059] The term "imide" refers to --C(O)NR.sup.98C(O)R.sup.99,
wherein R.sup.98 and R.sup.99 are each independently hydrogen, or a
substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl,
aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined
herein.
[0060] The term "imine" refers to --CR.sup.100(NR.sup.101) and
--N(CR.sup.100R.sup.101) groups, wherein R.sup.100 and R.sup.101
are each independently hydrogen or a substituted or unsubstituted
alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or
heterocyclylalkyl group as defined herein, with the proviso that
R.sup.100 and R.sup.101 are not both simultaneously hydrogen.
[0061] The term "nitro" as used herein refers to an --NO.sub.2
group.
[0062] The term "trifluoromethyl" as used herein refers to
--CF.sub.3.
[0063] The term "trifluoromethoxy" as used herein refers to
--OCF.sub.3.
[0064] The term "azido" refers to --N.sub.3.
[0065] The term "trialkyl ammonium" refers to a --N(alkyl).sub.3
group. A trialkylammonium group is positively charged and thus
typically has an associated anion, such as halogen anion.
[0066] The term "trifluoromethyldiazirido" refers to
##STR00001##
[0067] The term "isocyano" refers to --NC.
[0068] The term "isothiocyano" refers to --NCS.
[0069] The term "pentafluorosulfanyl" refers to --SF.sub.5.
[0070] As will be understood by one skilled in the art, for any and
all purposes, particularly in terms of providing a written
description, all ranges disclosed herein also encompass any and all
possible subranges and combinations of subranges thereof. Any
listed range can be easily recognized as sufficiently describing
and enabling the same range being broken down into at least equal
halves, thirds, quarters, fifths, tenths, etc. As a non-limiting
example, each range discussed herein can be readily broken down
into a lower third, middle third and upper third, etc. As will also
be understood by one skilled in the art all language such as "up
to," "at least," "greater than," "less than," and the like include
the number recited and refer to ranges which can be subsequently
broken down into subranges as discussed above. Finally, as will be
understood by one skilled in the art, a range includes each
individual member. Thus, for example, a group having 1-3 atoms
refers to groups having 1, 2, or 3 atoms. Similarly, a group having
1-5 atoms refers to groups having 1, 2, 3, 4, or 5 atoms, and so
forth.
[0071] Pharmaceutically acceptable salts of compounds described
herein are within the scope of the present technology and include
acid or base addition salts which retain the desired
pharmacological activity and is not biologically undesirable (e.g.,
the salt is not unduly toxic, allergenic, or irritating, and is
bioavailable). When the compound of the present technology has a
basic group, such as, for example, an amino group, pharmaceutically
acceptable salts can be formed with inorganic acids (such as
hydrochloric acid, hydroboric acid, nitric acid, sulfuric acid, and
phosphoric acid), organic acids (e.g., alginate, formic acid,
acetic acid, benzoic acid, gluconic acid, fumaric acid, oxalic
acid, tartaric acid, lactic acid, maleic acid, citric acid,
succinic acid, malic acid, methanesulfonic acid, benzenesulfonic
acid, naphthalene sulfonic acid, and p-toluenesulfonic acid) or
acidic amino acids (such as aspartic acid and glutamic acid). When
the compound of the present technology has an acidic group, such as
for example, a carboxylic acid group, it can form salts with
metals, such as alkali and earth alkali metals (e.g., Na.sup.+,
Li.sup.+, K.sup.+, Ca.sup.2+, Mg.sup.2+, Zn.sup.2+), ammonia or
organic amines (e.g. dicyclohexylamine, trimethylamine,
triethylamine, pyridine, picoline, ethanolamine, diethanolamine,
triethanolamine) or basic amino acids (e.g., arginine, lysine and
ornithine). Such salts can be prepared in situ during isolation and
purification of the compounds or by separately reacting the
purified compound in its free base or free acid form with a
suitable acid or base, respectively, and isolating the salt thus
formed.
[0072] Those of skill in the art will appreciate that compounds of
the present technology may exhibit the phenomena of tautomerism,
conformational isomerism, geometric isomerism and/or
stereoisomerism. As the formula drawings within the specification
and claims can represent only one of the possible tautomeric,
conformational isomeric, stereochemical or geometric isomeric
forms, it should be understood that the present technology
encompasses any tautomeric, conformational isomeric, stereochemical
and/or geometric isomeric forms of the compounds having one or more
of the utilities described herein, as well as mixtures of these
various different forms.
[0073] "Tautomers" refers to isomeric forms of a compound that are
in equilibrium with each other. The presence and concentrations of
the isomeric forms will depend on the environment the compound is
found in and may be different depending upon, for example, whether
the compound is a solid or is in an organic or aqueous solution.
For example, in aqueous solution, quinazolinones may exhibit the
following isomeric forms, which are referred to as tautomers of
each other:
##STR00002##
As another example, guanidines may exhibit the following isomeric
forms in protic organic solution, also referred to as tautomers of
each other:
##STR00003##
[0074] Because of the limits of representing compounds by
structural formulas, it is to be understood that all chemical
formulas of the compounds described herein represent all tautomeric
forms of compounds and are within the scope of the present
technology.
[0075] Stereoisomers of compounds (also known as optical isomers)
include all chiral, diastereomeric, and racemic forms of a
structure, unless the specific stereochemistry is expressly
indicated. Thus, compounds used in the present technology include
enriched or resolved optical isomers at any or all asymmetric atoms
as are apparent from the depictions. Both racemic and
diastereomeric mixtures, as well as the individual optical isomers
can be isolated or synthesized so as to be substantially free of
their enantiomeric or diastereomeric partners, and these
stereoisomers are all within the scope of the present
technology.
[0076] The compounds of the present technology may exist as
solvates, especially hydrates. Hydrates may form during manufacture
of the compounds or compositions comprising the compounds, or
hydrates may form over time due to the hygroscopic nature of the
compounds. Compounds of the present technology may exist as organic
solvates as well, including DMF, ether, and alcohol solvates among
others. The identification and preparation of any particular
solvate is within the skill of the ordinary artisan of synthetic
organic or medicinal chemistry.
[0077] Throughout this disclosure, various publications, patents
and published patent specifications are referenced by an
identifying citation. Also within this disclosure are Arabic
numerals referring to referenced citations, the full bibliographic
details of which are provided in sections within the Examples. The
disclosures of these publications, patents and published patent
specifications are hereby incorporated by reference into the
present disclosure to more fully describe the present
technology.
THE PRESENT TECHNOLOGY
[0078] In general, there is a need for radiotherapeutic compounds
that accumulate to a greater degree in tumors without unacceptable
uptake in normal non-target tissues and organs, as absorbed dose is
a function of the integral of cumulative activity. By targeting a
cellular marker on a neoplastic tissue, the radiotherapeutic can be
delivered to the tumor preferentially, thereby decreasing uptake in
non-target tissues. Furthermore, by including additional targeting
structures on the compound, the pharmacokinetics of the compound
can be altered. For example, using a blood-targeting moiety can
increase circulatory residence time, which has the effects of
increasing tumor perfusion and loading while reducing accumulation
of the radiotherapy compound in non-target tissues. Additionally,
although targeted radiotherapy has been practiced for some time
using macrocyclic complexes of radionuclides, the macrocycles
currently in use (e.g., DOTA) generally form complexes with many
radionuclide metals, such as actinium, radium, bismuth, astatine,
lutetium, and lead isotopes among others. Alpha-emitting
radionuclides can provide greater cytotoxic effects, and thus for
therapy are considered substantially more potent, than
beta-emitting radionuclides. The instability of currently known
macrocyclic-containing compounds can result in some dissociation of
the radionuclide from the macrocycle, and this results in a lack of
selectivity to targeted tissue, which also results in toxicity to
non-targeted tissue. Thus, in addition to the multiple targeting
domains, novel macrocyclic-containing compounds can be
incorporated, that display increased retention of the chelated
metal.
[0079] The present technology provides new compounds that overcome
the problems of traditional radiotherapy compounds, particularly
important when using alpha-emitters, accumulating the compound to a
greater degree in tumors without unacceptable uptake in normal
tissues and organs. The present technology also includes
macrocyclic complexes that are substantially more stable than those
of the conventional art, providing for the use of alpha-emitting
radionuclides instead of beta radionuclides. Thus, the compounds of
the present technology advantageously target cancer cells more
effectively, with substantially less toxicity to non-targeted
tissue than complexes of the art. Moreover, the new complexes can
advantageously be produced at room temperature, in contrast to
DOTA-type complexes, which generally require elevated temperatures
(e.g., at least 80.degree. C.) for complexation with the
radionuclide.
[0080] Thus, in one aspect of the present technology, a compound is
provided that includes a tumor-binding domain, an albumin-binding
domain, and a cytocidal or cytostatic therapeutic agent, where the
tumor-binding domain includes an active site that is distal to and
sterically unimpeded by the albumin-binding domain and the
therapeutic agent, and the relative affinity of the tumor-binding
domain and the albumin-binding domain differ in specific affinity
by a factor of at least 100 to about 10,000--such as 100, about
500, 1,000, about 1,500, about 2,000, about 3,000, about 4,000,
about 5,000, about 6,000, about 7,000, about 8,000, about 9,000,
about 10,000, or any range including and/or in between any two of
these values. The term "sterically unimpeded," as used herein,
refers to non-interference between the respective domains of the
compound, i.e., the tumor-binding domain and the albumin-binding
domain of the compound each having sufficient flexibility and
length such that each can bind to their cognate target, and where
the respective domains are spaced apart sufficiently such that one
domain does not physically occlude the binding site of the other,
or cause electrostatic effects that significantly reduce the
binding affinity of the other domain for its' target. The term will
be well-appreciated by those in the biology and chemistry fields,
such as evidenced by Rong et al., Molecular modeling of the
interactions of glutamate carboxypeptidase II with its potent
NAAG-based inhibitors, J Med Chem. 2002 Sep. 12; 45(19):4140-52,
which models the active site of PSMA and details numerous compounds
having affinity for it; see also Kim. J. K. et al. Synthesis and
Properties of a Sterically Unencumbered 6-Silanediol Amino Acid. J.
Org. Chem. 2012, 77, 2901-2906; Feng, L. et al. An Extremely Facile
Aza-Bergman Rearrangement of Sterically Unencumbered Acyclic
3-Aza-3-ene-1,5-diynes. J. Org. Chem. 2003, 68, 2234-2242; and
Friedrichsen, B. P. et al. Sterically Encumbered Functional Groups:
An Investigation of Endo versus Exo Phosphoryl Complexation Using
Proton and Phosphorus-31 NMR. J. Am. Chem. Soc. 1990, 112,
8931-8941.
[0081] In a related aspect, a compound is provided that is a
multi-targeted agent having a plurality of sterically unimpeded
targeting domains, where the compound includes a first targeting
domain that includes a blood-protein binding domain having specific
affinity for binding human serum albumin in the range of about 0.25
to 50 micromolar, and a second targeting domain that includes a
tumor-binding domain having specific affinity for a tumor
associated molecular target in the range of about 0.1 to 75
nanomolar; and a therapeutic domain that includes a cytocidal or
cytostatic therapeutic agent; wherein the relative affinities of
the first and second targeting domains differ in specific affinity
by a factor of at least 100 to about 10,000--such as 100, about
500, 1,000, about 1,500, about 2,000, about 3,000, about 4,000,
about 5,000, about 6,000, about 7,000, about 8,000, about 9,000,
about 10,000, or any range including and/or in between any two of
these values.
[0082] The first targeting domain that includes the blood-protein
binding domain may have a specific affinity for binding human serum
albumin of about 0.25 micromolar, about 0.30 micromolar, about 0.35
micromolar, about 0.40 micromolar, about 0.45 micromolar, about
0.50 micromolar, about 0.55 micromolar, about 0.60 micromolar,
about 0.65 micromolar, about 0.70 micromolar, about 0.75
micromolar, about 0.80 micromolar, about 0.85 micromolar, about
0.90 micromolar, about 0.95 micromolar, about 1 micromolar, about 2
micromolar, about 3 micromolar, about 4 micromolar, about 5
micromolar, about 10 micromolar, about 15 micromolar, about 20
micromolar, about 25 micromolar, about 30 micromolar, about 35
micromolar, about 40 micromolar, about 45 micromolar, about 50
micromolar, or any range including and/or in between any two of
these values. The second targeting domain that includes the
tumor-binding domain may have a specific affinity for the tumor
associated molecular target of about 0.1 nanomolar, about 0.15
nanomolar, about 0.20 nanomolar, about 0.25 nanomolar, about 0.30
nanomolar, about 0.35 nanomolar, about 0.40 nanomolar, about 0.45
nanomolar, about 0.50 nanomolar, about 0.55 nanomolar, about 0.60
nanomolar, about 0.65 nanomolar, about 0.70 nanomolar, about 0.75
nanomolar, about 0.80 nanomolar, about 0.85 nanomolar, about 0.90
nanomolar, about 0.95 nanomolar, about 1 nanomolar, about 2
nanomolar, about 3 nanomolar, about 4 nanomolar, about 5 nanomolar,
about 10 nanomolar, about 15 nanomolar, about 20 nanomolar, about
25 nanomolar, about 30 nanomolar, about 35 nanomolar, about 40
nanomolar, about 45 nanomolar, about 50 nanomolar, about 55
nanomolar, about 60 nanomolar, about 65 nanomolar, about 70
nanomolar, about 75 nanomolar, or any range including and/or in
between any two of these values.
[0083] In a further related aspect, a compound is provided that is
a trefoil construct, where the trefoil construct includes a tumor
recognition moiety, an albumin binding moiety, and a radionuclide
moiety that does not participate strongly in either tumor target
recognition or albumin binding.
[0084] The tumor-binding domain (e.g., the tumor recognition
moiety) includes a moiety capable of recognizing or interacting
with a molecular target on the surface of tumor cells. Such
molecular targets include cell surface proteins such as receptors,
enzymes, and antigens. For example, the molecular target may be a
receptor, an enzyme, and/or an antigen expressed on a tumor cell
surface (such as a tumor-specific cell surface protein) capable of
interacting with the tumor-binding domain. An example of such a
tumor-binding domain is the glutamate-urea-lysine motif recognized
by prostate specific membrane antigen (PSMA) which is expressed on
the surface of most prostate cancer cells. Another example is
edotreotide, recognized by somatostatin receptors expressed on the
surface of many neuroendocrine cancers. Thus, the tumor-binding
domain (e.g., the tumor recognition moiety) of any aspect and
embodiment herein is capable of binding to a tumor associated
molecular target that includes one or more of: a tumor associated
molecular target that is a tumor-specific cell surface protein or
other marker such as prostate specific membrane antigen (PSMA),
somatostatin peptide receptor-2 (SSTR2), alphavbeta3
(.alpha.v.beta.3), alphavbeta6, a gastrin-releasing peptide
receptor, a seprase, fibroblast activation protein alpha
(FAP-alpha), an incretin receptor, a glucose-dependent
insulinotropic polypeptide receptor, VIP-1, NPY, a folate receptor,
LHRH, a neuronal transporter (e.g., noradrenaline transporter
(NET)), EGFR, HER-2, VGFR, MUC-1, CEA, MUC-4, ED2, TF-antigen, an
endothelial specific marker, neuropeptide Y, uPAR, TAG-72, a
claudin, a CCK analog, VIP, bombesin, VEGFR, a tumor-specific cell
surface protein, GLP-1, CXCR4, Hepsin, TMPRSS2, a caspace, cMET, or
an overexpressed peptide receptor. The preceeding are simply
representative tumor associated molecular targets and for which
detailed structural information exists for both the target and
compounds that bind it. The various antibodies, peptides and
compounds that display specific affinity for these particular
cellular targets are widely described in the scientific literature,
and can be adapted to the instant invention as tumor-binding
domains. The tumor-binding domain (e.g., the tumor recognition
moiety) of any aspect and embodiment herein is capable of binding
to the tumor associated molecular target with at least moderate
adffinity, to high affinity (e.g., the equilibrium binding constant
(K.sub.D) ranging from about 10{circumflex over ( )}-8M to about
10{circumflex over ( )}-10M). Currently preferred tumor-binding
that target and bind to the active site of PSMA, include for
example, a glutamate-ureido-amino acid sequence, a
glutamate-urea-lysine sequence with or without an aromatic
substituent at the epsilon amine of lysine, or any derivative
thereof that can bind the active site of PSMA with moderate to high
affinity. Exemplary structures are provided herein, however other
regions of PSMA can be targeted, and these are interchangeable with
the PSMA tumor-binding domains in the compounds detailed
herein.
[0085] The blood-protein binding domain (e.g., the albumin-binding
domain; the albumin-binding moiety) plays a role in modulating the
rate of blood plasma clearance of the compounds in a subject,
thereby increasing circulation time and compartmentalizing the
cytotoxic action of cytotoxin-containing domain and/or imaging
capability of the imaging agent-containing domain in the plasma
space instead of normal organs and tissues that may express
antigen. Without being bound by theory, this component of the
compound is believed to interact reversibly with serum proteins,
such as albumin and/or cellular elements. The affinity of this
blood-protein binding domain (e.g., the albumin-binding domain; the
albumin-binding moiety) for plasma or cellular components of the
blood may be configured to affect the residence time of the
compounds in the blood pool of a subject. In any embodiment herein,
the blood-protein binding domain (e.g., the albumin-binding domain;
the albumin-binding moiety) may be configured so that it binds
reversibly or non-reversibly with albumin when in blood plasma.
[0086] By way of example, the blood-protein binding domain of any
aspect or embodiment herein may include a short-chain fatty acid,
medium-chain chain fatty acid, a long-chain fatty acid, myristic
acid, a substituted or unsubstituted indole-2-carboxylic acid, a
substituted or unsubstituted thioamide, a substituted or
unsubstituted 4-oxo-4-(5,6,7,8-tetrahydronaphthalen-2-yl)butanoic
acid, a substituted or unsubstituted naphthalene acylsulfonamide, a
substituted or unsubstituted diphenylcyclohexanol phosphate ester,
a substituted or unsubstituted 4-iodophenylalkanoic acid, a
substituted or unsubstituted 3-(4-iodophenyl)propionic acid, a
substituted or unsubstituted 2-(4-iodophenyl)acetic acid, or a
substituted or unsubstituted 4-(4-iodophenyl)butanoic acid. Certain
representative examples of moieties that bind the blood protein
albumin, that may be included in any embodiment herein include one
or more of the following:
##STR00004##
[0087] The cytocidal or cytostatic therapeutic agent of any aspect
and embodiment herein may include a toxin, a venom, a metabolic
poison, a chemotherapeutic agent, an auger electron-emitting
radionuclide, a beta-emitting radionuclide, or an alpha-emitting
radionuclide. A moiety that includes a radionuclide is also
referred to herein as a "radionuclide moiety." The cytocidal or
cytostatic therapeutic agent of any aspect and embodiment herein
may include a covalently conjugated chelating agent or a polyaza
polycarboxylic macrocycle (collectively, "chelators") which may
further chelate a metal ion; the radionuclide moiety may include a
covalently conjugated chelating agent or a polyaza polycarboxylic
macrocycle chelating a radionuclide. Such chelated metal ions may
provide that the compounds of the present technology may be used
in, e.g., magnetic resonance imaging, luminescence imaging,
radiotherapy, or a combination of any two or more thereof. The
metal ion of any aspect and embodiment herein may be a
radionuclide, such as .sup.177Lu.sup.3+, .sup.175Lu.sup.3+,
.sup.45Sc.sup.3+, .sup.66Ga.sup.3+, .sup.67Ga.sup.3+,
.sup.68Ga.sup.3+, .sup.69Ga.sup.3+, .sup.71Ga.sup.3+,
.sup.90Y.sup.3+, .sup.89Y.sup.3+, .sup.86Y.sup.3+,
.sup.89Zr.sup.4+, .sup.90Y.sup.3+, .sup.99mTc+.sup.1,
.sup.111In.sup.3+, .sup.113In.sup.3+, .sup.115In.sup.3+,
.sup.139La.sup.3+, .sup.136Ce.sup.3+, .sup.138Ce.sup.3+,
.sup.140Ce.sup.3+, .sup.142Ce.sup.3+, .sup.151Eu.sup.3+,
.sup.153Eu.sup.3+, .sup.152Dy.sup.3+, .sup.149Tb.sup.3+,
.sup.159Tb.sup.3+, .sup.154Gd.sup.3+, .sup.155Gd.sup.3+,
.sup.156Gd.sup.3+, .sup.157Gd.sup.3+, .sup.158Gd.sup.3+,
.sup.160Gd.sup.3+, .sup.188Re+.sup.1, .sup.186Re+.sup.1,
.sup.213Bi.sup.3+, .sup.211At.sup.+, .sup.217At.sup.+,
.sup.227Th.sup.4+, .sup.226Th.sup.4+, .sup.225Ac.sup.3+,
.sup.233Ra.sup.2+, .sup.152Dy.sup.3+, .sup.213Bi.sup.3+,
.sup.212Bi.sup.3+, .sup.211Bi.sup.3+, .sup.212Pb.sup.2+,
.sup.212Pb.sup.4+, .sup.255Fm.sup.3+, or uranium-230. For example,
the metal ion may be an alpha-emitting radionuclide such as
.sup.213Bi.sup.3+, .sup.211At.sup.+, .sup.225Ac.sup.3+,
.sup.152Dy.sup.3+, .sup.212Bi.sup.3+, .sup.211Bi.sup.3+,
.sup.217At.sup.+, .sup.227Th.sup.4+, .sup.226Th.sup.4+,
.sup.233Ra.sup.2+, .sup.212Pb.sup.2+, and .sup.212Pb.sup.4+; for
example, the metal ion may be a beta-emitting radionuclide such as
.sup.177Lu.sup.3+, .sup.90Y.sup.3+, .sup.188Re+.sup.1, and
.sup.186Re+.sup.1. The radionuclide of any embodiment herein may be
a therapeutic radionuclide, a diagnostic radionuclide, or both.
[0088] Chelating agents and polyaza polycarboxylic macrocycles
useful in any embodiment of the present technology include, but are
not limited to, a covalently conjugated substituted or
unsubstituted member of the following group: [0089]
1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), [0090]
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA),
[0091] p-SCN-Bn-DOTA (also known as 2B-DOTA-NCS), [0092] PIP-DOTA,
[0093] diethylenetriaminepentaacetic acid (DTPA), [0094] PIP-DTPA,
[0095] AZEP-DTPA, [0096] ethylenediamine tetraacetic acid (EDTA),
[0097] triethylenetetraamine-N,N,N',N'',N''',N'''-hexa-acetic acid
(TTHA), [0098]
7-[2-(bis-carboxymethylamino)-ethyl]-4,10-bis-carboxymethyl-1,4,7,10-tetr-
aaza-cyclododec-1-yl-acetic acid (DEPA), [0099]
2,2',2''-(10-(2-(bis(carboxymethyl)amino)-5-(4-isothiocyanatophenyl)
pentyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid
(3p-C-DEPA-NCS), [0100] NETA, [0101]
{4-carboxymethyl-7-[2-(carboxymethylamino)-ethyl]-perhydro-1,4,7-triazoni-
n-1-yl}-acetic acid (NPTA), [0102]
diacetylpyridinebis(benzoylhydrazone), [0103]
1,4,7,10,13,16-hexaazacyclooctadecane-N,N',N'',N''',N'''',N'''''-h-
exaacetic acid (HEHA), octadentate terephthalamide ligands, [0104]
siderophores, [0105]
2,2'-(4-(2-(bis(carboxymethyl)amino)-5-(4-isothiocyanatophenyl)pentyl)-10-
-(2-(bis(carboxymethyl)amino)ethyl)-1,4,7,10-tetraazacyclododecane-1,7-diy-
l)diacetic acid, [0106]
N,N-bis[(6-carboxy-2-pyridil)methyl]-4,13-diaza-18-crown-6
(H.sub.2macropa), [0107]
6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclo-
octadecan-7-yl)methyl)-4-isothiocyanatopicolinic acid
(macropa-NCS), and [0108]
3,9-carboxymethyl-6-(2-methoxy-5-isothiocyanatophenyl)carboxymethy-
l-3,6,9,15-tetraazabicyclo-[9.3.1]pentadeca-1(15),11,13-triene.
Certain members of this exemplary group are illustrated below.
##STR00005## ##STR00006##
[0109] It is to be understood that a "covalently conjugated"
chelating agent or a "covalently conjugated" polyaza polycarboxylic
macrocycle means a chelating agent or polyaza polycarboxylic
macrocycle (such as those listed above) wherein one or more bonds
to a hydrogen atom contained therein are replaced by a bond to an
atom of the remainder of the compound of the present technology or
a pi bond between two atoms is replaced by a bond from one of the
two atoms to the compound of the present technology and the other
of the two atoms includes a new bond, e.g. to a hydrogen (such as
reaction of an --NCS group in a chelating agent to provide the
covalently conjugated chelating agent).
[0110] The present technology also provides compositions and
medicaments comprising anyone of the aspects and embodiments of the
compounds of the present technology and a pharmaceutically
acceptable carrier or one or more excipients or fillers
(collectively referred to as "pharmaceutically acceptable carrier"
unless otherwise specified). The compositions may be used in the
methods and treatments described herein. The present technology
also provides pharmaceutical compositions including a
pharmaceutically acceptable carrier and an effective amount of a
compound of any one of the aspects and embodiments of the compounds
of the present technology for imaging and/or treating a condition;
and where the condition may include a glioma, a breast cancer, an
adrenal cortical cancer, a cervical carcinoma, a vulvar carcinoma,
an endometrial carcinoma, a primary ovarian carcinoma, a metastatic
ovarian carcinoma, a non-small cell lung cancer, a small cell lung
cancer, a bladder cancer, a colon cancer, a primary, gastric
adenocarcinoma, a primary colorectal adenocarcinoma, a renal cell
carcinoma, and/or a prostate cancer. For example, such conditions
may include a mammalian tissue overexpressing PSMA, such as a
cancer expressing PSMA (including cancer tissues, cancer related
neo-vasculature, or a combination thereof), Crohn's disease, or
IBD.
[0111] In a further related aspect, an imaging method is provided
that includes administering a compound of any one of the aspects
and embodiments of the compounds of the present technology (e.g.,
such as administering an effective amount) or administering a
pharmaceutical composition comprising an effective amount of a
compound of any one of the aspects and embodiments of the compounds
of the present technology to a subject and, subsequent to the
administering, detecting positron emission, detecting gamma rays
from positron emission and annihilation (such as by positron
emission tomography), and/or detecting Cerenkov radiation due to
positron emission (such as by Cerenkov luminescene imaging). In any
embodiment of the imaging method, the subject may be suspected of
suffering from a condition that includes a glioma, a breast cancer,
an adrenal cortical cancer, a cervical carcinoma, a vulvar
carcinoma, an endometrial carcinoma, a primary ovarian carcinoma, a
metastatic ovarian carcinoma, a non-small cell lung cancer, a small
cell lung cancer, a bladder cancer, a colon cancer, a primary,
gastric adenocarcinoma, a primary colorectal adenocarcinoma, a
renal cell carcinoma, a prostate cancer, a mammalian tissue
overexpressing PSMA, such as a cancer expressing PSMA (including
cancer tissues, cancer related neo-vasculature, or a combination
thereof), Crohn's disease, or IBD. The detecting step may occur
during a surgical procedure on a subject, e.g., to remove a
mammalian tissue overexpressing PSMA. The detecting step may
include use of a handheld device to perform the detecting step. For
example, Cerenkov luminescene images may be acquired by detecting
the Cerenkov light using ultra-high-sensitivity optical cameras
such as electron-multiplying charge-coupled device (EMCCD)
cameras.
[0112] In any of the above embodiments, the effective amount may be
determined in relation to a subject. "Effective amount" refers to
the amount of a compound or composition required to produce a
desired effect. One non-limiting example of an effective amount
includes amounts or dosages that yield acceptable toxicity and
bioavailability levels for therapeutic (pharmaceutical) use
including, but not limited to, the treatment of e.g., a glioma, a
breast cancer, an adrenal cortical cancer, a cervical carcinoma, a
vulvar carcinoma, an endometrial carcinoma, a primary ovarian
carcinoma, a metastatic ovarian carcinoma, a non-small cell lung
cancer, a small cell lung cancer, a bladder cancer, a colon cancer,
a primary, gastric adenocarcinoma, a primary colorectal
adenocarcinoma, a renal cell carcinoma, and a prostate cancer.
Another example of an effective amount includes amounts or dosages
that are capable of reducing symptoms associated with e.g., a
glioma, a breast cancer, an adrenal cortical cancer, a cervical
carcinoma, a vulvar carcinoma, an endometrial carcinoma, a primary
ovarian carcinoma, a metastatic ovarian carcinoma, a non-small cell
lung cancer, a small cell lung cancer, a bladder cancer, a colon
cancer, a primary, gastric adenocarcinoma, a primary colorectal
adenocarcinoma, a renal cell carcinoma, or a prostate cancer, such
as, for example, reduction in proliferation and/or metastasis. An
effective amount of a compound of the present technology may
include an amount sufficient to enable detection of binding of the
compound to a target of interest including, but not limited to, one
or more of a glioma, a breast cancer, an adrenal cortical cancer, a
cervical carcinoma, a vulvar carcinoma, an endometrial carcinoma, a
primary ovarian carcinoma, a metastatic ovarian carcinoma, a
non-small cell lung cancer, a small cell lung cancer, a bladder
cancer, a colon cancer, a primary, gastric adenocarcinoma, a
primary colorectal adenocarcinoma, a renal cell carcinoma, or a
prostate cancer (such as castration resistant prostate cancer).
Another example of an effective amount includes amounts or dosages
that are capable of providing a detectable gamma ray emission from
positron emission and annihilation (above background) in a subject
with a tissue overexpressing PSMA, such as, for example,
statistically significant emission above background. Another
example of an effective amount includes amounts or dosages that are
capable of providing a detectable Cerenkov radiation emission due
to positron emission above background) in a subject with a tissue
overexpressing PSMA, such as, for example, statistically
significant emission above background. The effective amount may be
from about 0.01 g to about 1 mg of the compound per gram of the
composition, and preferably from about 0.1 g to about 500 g of the
compound per gram of the composition.
[0113] As used herein, a "subject" or "patient" is a mammal, such
as a cat, dog, rodent or primate. Typically the subject is a human,
and, preferably, a human suffering from or suspected of suffering
from a glioma, a breast cancer, an adrenal cortical cancer, a
cervical carcinoma, a vulvar carcinoma, an endometrial carcinoma, a
primary ovarian carcinoma, a metastatic ovarian carcinoma, a
non-small cell lung cancer, a small cell lung cancer, a bladder
cancer, a colon cancer, a primary, gastric adenocarcinoma, a
primary colorectal adenocarcinoma, a renal cell carcinoma, or a
prostate cancer. The term "subject" and "patient" can be used
interchangeably.
[0114] In particular, the effective amount of a compound of any
embodiment herein for treating a cancer and/or a mammalian tissue
overexpressing PSMA may be from about 0.1 .mu.g to about 50 .mu.g
per kilogram of the mass of the subject. Thus, for treating a
cancer (e.g., a glioma, a breast cancer, an adrenal cortical
cancer, a cervical carcinoma, a vulvar carcinoma, an endometrial
carcinoma, a primary ovarian carcinoma, a metastatic ovarian
carcinoma, a non-small cell lung cancer, a small cell lung cancer,
a bladder cancer, a colon cancer, a primary, gastric
adenocarcinoma, a primary colorectal adenocarcinoma, a renal cell
carcinoma, a prostate cancer, and/or a castration resistant
prostate cancer) and/or a mammalian tissue overexpressing PSMA; the
effective amount of a compound of any embodiment described herein
may be about 0.1 .mu.g/kg, about 0.2 .mu.g/kg, about 0.3 .mu.g/kg,
about 0.4 .mu.g/kg, about 0.5 .mu.g/kg, about 0.6 .mu.g/kg, about
0.7 .mu.g/kg, about 0.8 .mu.g/kg, about 0.9 .mu.g/kg, about 1
.mu.g/kg, about 2 .mu.g/kg, about 3 .mu.g/kg, about 4 .mu.g/kg,
about 5 .mu.g/kg, about 6 .mu.g/kg, about 7 .mu.g/kg, about 8
.mu.g/kg, about 9 .mu.g/kg, about 10 .mu.g/kg, about 11 .mu.g/kg,
about 12 .mu.g/kg, about 13 .mu.g/kg, about 14 .mu.g/kg, about 15
.mu.g/kg, about 16 .mu.g/kg, about 17 .mu.g/kg, about 18 .mu.g/kg,
about 19 .mu.g/kg, about 20 .mu.g/kg, about 22 .mu.g/kg, about 24
.mu.g/kg, about 26 .mu.g/kg, about 28 .mu.g/kg, about 30 .mu.g/kg,
about 32 .mu.g/kg, about 34 .mu.g/kg, about 36 .mu.g/kg, about 38
.mu.g/kg, about 40 .mu.g/kg, about 42 .mu.g/kg, about 44 .mu.g/kg,
about 46 .mu.g/kg, about 48 .mu.g/kg, about 50 .mu.g/kg, or any
range including and/or in between any two of these values.
[0115] In particular, the effective amount of a compound of any
embodiment herein for imaging a cancer and/or a mammalian tissue
overexpressing PSMA may be from about 0.1 .mu.g to about 50 .mu.g
per kilogram of the mass of the subject. Thus, for treating a
cancer (e.g., a glioma, a breast cancer, an adrenal cortical
cancer, a cervical carcinoma, a vulvar carcinoma, an endometrial
carcinoma, a primary ovarian carcinoma, a metastatic ovarian
carcinoma, a non-small cell lung cancer, a small cell lung cancer,
a bladder cancer, a colon cancer, a primary, gastric
adenocarcinoma, a primary colorectal adenocarcinoma, a renal cell
carcinoma, a prostate cancer, and/or a castration resistant
prostate cancer) and/or a mammalian tissue overexpressing PSMA; the
effective amount of a compound of any embodiment described herein
may be about 0.1 .mu.g/kg, about 0.2 .mu.g/kg, about 0.3 .mu.g/kg,
about 0.4 .mu.g/kg, about 0.5 .mu.g/kg, about 0.6 .mu.g/kg, about
0.7 .mu.g/kg, about 0.8 .mu.g/kg, about 0.9 .mu.g/kg, about 1
.mu.g/kg, about 2 .mu.g/kg, about 3 .mu.g/kg, about 4 .mu.g/kg,
about 5 .mu.g/kg, about 6 .mu.g/kg, about 7 .mu.g/kg, about 8
.mu.g/kg, about 9 .mu.g/kg, about 10 .mu.g/kg, about 11 .mu.g/kg,
about 12 .mu.g/kg, about 13 .mu.g/kg, about 14 .mu.g/kg, about 15
.mu.g/kg, about 16 .mu.g/kg, about 17 .mu.g/kg, about 18 .mu.g/kg,
about 19 .mu.g/kg, about 20 .mu.g/kg, about 22 .mu.g/kg, about 24
.mu.g/kg, about 26 .mu.g/kg, about 28 .mu.g/kg, about 30 .mu.g/kg,
about 32 .mu.g/kg, about 34 .mu.g/kg, about 36 .mu.g/kg, about 38
.mu.g/kg, about 40 .mu.g/kg, about 42 .mu.g/kg, about 44 .mu.g/kg,
about 46 .mu.g/kg, about 48 .mu.g/kg, about 50 .mu.g/kg, or any
range including and/or in between any two of these values.
[0116] The compounds of the present technology may also be
administered to a patient along with other conventional imaging
agents that may be useful in the imaging and/or treatment of a
glioma, a breast cancer, an adrenal cortical cancer, a cervical
carcinoma, a vulvar carcinoma, an endometrial carcinoma, a primary
ovarian carcinoma, a metastatic ovarian carcinoma, a non-small cell
lung cancer, a small cell lung cancer, a bladder cancer, a colon
cancer, a primary, gastric adenocarcinoma, a primary colorectal
adenocarcinoma, a renal cell carcinoma, a prostate cancer, or a
mammalian tissue overexpressing PSMA. Such mammalian tissues
include, but are not limited to, a cancer expressing PSMA
(including cancer tissues, cancer related neo-vasculature, or a
combination thereof), Crohn's disease, or IBD. Thus, a
pharmaceutical composition and/or method of the present technology
may further include an imaging agent different than the compounds
of Formulas I-III; a pharmaceutical composition and/or method of
the present technology may include an treatment agent different
than the compounds of the present technology; a pharmaceutical
composition and/or method of the present technology may further
include an imaging agent according to any embodiment of a compound
of the present technology and therapeutic agent that is also
according to any embodiment of a compound of the present
technology. It may be that the compound according to the present
technology is both a therapeutic agent and an imaging agent. The
administration may include oral administration, parenteral
administration, or nasal administration. In any of these
embodiments, the administration may include subcutaneous
injections, intravenous injections, intraperitoneal injections, or
intramuscular injections. In any of these embodiments, the
administration may include oral administration. The methods of the
present technology may also include administering, either
sequentially or in combination with one or more compounds of the
present technology, a conventional imaging agent in an amount that
can potentially or synergistically be effective for the imaging of
a mammalian tissue overexpressing PSMA.
[0117] In any of the embodiments of the present technology
described herein, the pharmaceutical composition may be packaged in
unit dosage form. The unit dosage form is effective in treating a
glioma, a breast cancer, an adrenal cortical cancer, a cervical
carcinoma, a vulvar carcinoma, an endometrial carcinoma, a primary
ovarian carcinoma, a metastatic ovarian carcinoma, a non-small cell
lung cancer, a small cell lung cancer, a bladder cancer, a colon
cancer, a primary, gastric adenocarcinoma, a primary colorectal
adenocarcinoma, a renal cell carcinoma, and/or a prostate cancer.
Generally, a unit dosage including a compound of the present
technology will vary depending on patient considerations. Such
considerations include, for example, age, protocol, condition, sex,
extent of disease, contraindications, concomitant therapies and the
like. An exemplary unit dosage based on these considerations may
also be adjusted or modified by a physician skilled in the art. For
example, a unit dosage for a patient comprising a compound of the
present technology may vary from 1.times.10.sup.-4 g/kg to 1 g/kg,
preferably, 1.times.10.sup.-3 g/kg to 1.0 g/kg. Dosage of a
compound of the present technology may also vary from 0.01 mg/kg to
100 mg/kg or, preferably, from 0.1 mg/kg to 10 mg/kg. Suitable unit
dosage forms, include, but are not limited to powders, tablets,
pills, capsules, lozenges. suppositories. patches. nasal sprays,
injectibles, implantable sustained-release formulations,
rnucoadherent films, topical varnishes, lipid complexes, etc.
[0118] The pharmaceutical compositions may be prepared by mixing
one or more compounds of the present technology with
pharmaceutically acceptable carriers, excipients, binders, diluents
or the like to prevent and treat disorders associated with cancer
(e.g., a glioma, a breast cancer, an adrenal cortical cancer, a
cervical carcinoma, a vulvar carcinoma, an endometrial carcinoma, a
primary ovarian carcinoma, a metastatic ovarian carcinoma, a
non-small cell lung cancer, a small cell lung cancer, a bladder
cancer, a colon cancer, a primary, gastric adenocarcinoma, a
primary colorectal adenocarcinoma, a renal cell carcinoma, and a
prostate cancer). The compounds and compositions described herein
may be used to prepare formulations and medicaments that treat
e.g., a glioma, a breast cancer, an adrenal cortical cancer, a
cervical carcinoma, a vulvar carcinoma, an endometrial carcinoma, a
primary ovarian carcinoma, a metastatic ovarian carcinoma, a
non-small cell lung cancer, a small cell lung cancer, a bladder
cancer, a colon cancer, a primary, gastric adenocarcinoma, a
primary colorectal adenocarcinoma, a renal cell carcinoma, and a
prostate cancer. Such compositions may be in the form of, for
example, granules, powders, tablets, capsules, syrup,
suppositories, injections, emulsions, elixirs, suspensions or
solutions. The instant compositions may be formulated for various
routes of administration, for example, by oral, parenteral,
topical, rectal, nasal, vaginal administration, or via implanted
reservoir. Parenteral or systemic administration includes, but is
not limited to, subcutaneous, intravenous, intraperitoneal, and
intramuscular, injections. The following dosage forms are given by
way of example and should not be construed as limiting the instant
present technology.
[0119] For oral, buccal, and sublingual administration, powders,
suspensions, granules, tablets, pills, capsules, gelcaps, and
caplets are acceptable as solid dosage forms. These can be
prepared, for example, by mixing one or more compounds of the
instant present technology, or pharmaceutically acceptable salts or
tautomers thereof, with at least one additive such as a starch or
other additive. Suitable additives are sucrose, lactose, cellulose
sugar, mannitol, maltitol, dextran, starch, agar, alginates,
chitins, chitosans, pectins, tragacanth gum, gum arabic, gelatins,
collagens, casein, albumin, synthetic or semi-synthetic polymers or
glycerides. Optionally, oral dosage forms can contain other
ingredients to aid in administration, such as an inactive diluent,
or lubricants such as magnesium stearate, or preservatives such as
paraben or sorbic acid, or anti-oxidants such as ascorbic acid,
tocopherol or cysteine, a disintegrating agent, binders,
thickeners, buffers, sweeteners, flavoring agents or perfuming
agents. Tablets and pills may be further treated with suitable
coating materials known in the art.
[0120] Liquid dosage forms for oral administration may be in the
form of pharmaceutically acceptable emulsions, syrups, elixirs,
suspensions, and solutions, which may contain an inactive diluent,
such as water. Pharmaceutical formulations and medicaments may be
prepared as liquid suspensions or solutions using a sterile liquid,
such as, but not limited to, an oil, water, an alcohol, and
combinations of these. Pharmaceutically suitable surfactants,
suspending agents, emulsifying agents, may be added for oral or
parenteral administration.
[0121] As noted above, suspensions may include oils. Such oils
include, but are not limited to, peanut oil, sesame oil, cottonseed
oil, corn oil and olive oil. Suspension preparation may also
contain esters of fatty acids such as ethyl oleate, isopropyl
myristate, fatty acid glycerides and acetylated fatty acid
glycerides. Suspension formulations may include alcohols, such as,
but not limited to, ethanol, isopropyl alcohol, hexadecyl alcohol,
glycerol and propylene glycol. Ethers, such as but not limited to,
poly(ethyleneglycol), petroleum hydrocarbons such as mineral oil
and petrolatum; and water may also be used in suspension
formulations.
[0122] Injectable dosage forms generally include aqueous
suspensions or oil suspensions which may be prepared using a
suitable dispersant or wetting agent and a suspending agent.
Injectable forms may be in solution phase or in the form of a
suspension, which is prepared with a solvent or diluent. Acceptable
solvents or vehicles include sterilized water, Ringer's solution,
or an isotonic aqueous saline solution. Alternatively, sterile oils
may be employed as solvents or suspending agents. Typically, the
oil or fatty acid is non-volatile, including natural or synthetic
oils, fatty acids, mono-, di- or tri-glycerides.
[0123] For injection, the pharmaceutical formulation and/or
medicament may be a powder suitable for reconstitution with an
appropriate solution as described above. Examples of these include,
but are not limited to, freeze dried, rotary dried or spray dried
powders, amorphous powders, granules, precipitates, or
particulates. For injection, the formulations may optionally
contain stabilizers, pH modifiers, surfactants, bioavailability
modifiers and combinations of these.
[0124] Compounds of the present technology may be administered to
the lungs by inhalation through the nose or mouth. Suitable
pharmaceutical formulations for inhalation include solutions,
sprays, dry powders, or aerosols containing any appropriate
solvents and optionally other compounds such as, but not limited
to, stabilizers, antimicrobial agents, antioxidants, pH modifiers,
surfactants, bioavailability modifiers and combinations of these.
The carriers and stabilizers vary with the requirements of the
particular compound, but typically include nonionic surfactants
(Tweens, Pluronics, or polyethylene glycol), innocuous proteins
like serum albumin, sorbitan esters, oleic acid, lecithin, amino
acids such as glycine, buffers, salts, sugars or sugar alcohols.
Aqueous and nonaqueous (e.g., in a fluorocarbon propellant)
aerosols are typically used for delivery of compounds of the
present technology by inhalation.
[0125] Besides those representative dosage forms described above,
pharmaceutically acceptable excipients and carriers are generally
known to those skilled in the art and are thus included in the
instant present technology. Such excipients and carriers are
described, for example, in "Remingtons Pharmaceutical Sciences"
Mack Pub. Co., New Jersey (1991), which is incorporated herein by
reference. The instant compositions may also include, for example,
micelles or liposomes, or some other encapsulated form.
[0126] Specific dosages may be adjusted depending on conditions of
disease, the age, body weight, general health conditions, sex, and
diet of the subject, dose intervals, administration routes,
excretion rate, and combinations of drugs. Any of the above dosage
forms containing effective amounts are well within the bounds of
routine experimentation and therefore, well within the scope of the
instant present technology.
[0127] Various assays and model systems can be readily employed to
determine the therapeutic effectiveness of the treatment according
to the present technology.
[0128] For the indicated condition, test subjects will exhibit a
10%, 20%, 30%, 50% or greater reduction, up to a 75-90%, or 95% or
greater, reduction, in one or more symptom(s) caused by, or
associated with, the disorder in the subject, compared to
placebo-treated or other suitable control subjects.
[0129] The present technology further provides a method of
achieving an in vivo tissue distribution of a radiotherapeutic in a
mammalian subject in which a ratio of tumor activity to kidney
activity of 1 or greater is observed within about 4 hours to about
24 hours of administration of the radiotherapeutic to the mammalian
subject. Such a method includes administering to the mammalian
subject the radiotherapeutic, where the radiotherapeutic comprises
a first moiety that targets prostate specific membrane antigen
("PSMA"), a second moiety that bears a radionuclide, and a third
moiety that has an affinity for serum albumin, the first moiety
being separated from the second moiety by a first covalent linker
and the third moiety being separated from the second moiety by a
second covalent linker. The separation between the first and second
moieties (on the basis of a contiguous atom count associated with
the first covalent linker) is from about 8 atoms to about 40 atoms,
and the separation between the third moiety and the first and
second moieties (on the basis of a contiguous atom count associated
with the second covalent linker) is from about 10 atoms to about
100 atoms.
[0130] The method may include obtaining an image of the mammalian
subject about 4 hours to about 24 hours after administration of the
radiotherapeutic; thus, obtaining an image after administration of
the radiotherapeutic may occur after about 4 hours, about 5 hours,
about 6 hours, about 7 hours, about 8 hours, about 9 hours, about
10 hours, about 11 hours, about 12 hours, about 13 hours, about 14
hours, about 15 hours, about 16 hours, about 17 hours, about 18
hours, about 19 hours, about 20 hours, about 21 hours, about 22
hours, about 23 hours, about 24 hours, or any range including
and/or in between any two of these values. The ratio of tumor
activity to kidney activity of 1 or greater may persist up to about
24 hours after administration of the radiotherapeutic. In any
embodiment herein of the method, it may be that substantially no
radionuclide activity is observed in salivary glands of the
mammalian subject about 24 hours to about 48 hours after
administration of the radiotherapeutic. In any embodiment herein of
the method, it may be that the contiguous atom count associated
with the first covalent linker ranges from about 10 atoms to about
30 atoms. In any embodiment herein of the method, it may be that
the contiguous atom count associated with the second covalent
linker ranges from about 15 atoms to about 40 atoms. In any
embodiment herein of the method, it may be that the administration
comprises intravenous administration.
[0131] The examples herein are provided to illustrate advantages of
the present technology and to further assist a person of ordinary
skill in the art with preparing or using the compounds of the
present technology or salts, pharmaceutical compositions,
derivatives, prodrugs, or tautomeric forms thereof. The examples
herein are also presented in order to more fully illustrate the
preferred aspects of the present technology. The examples should in
no way be construed as limiting the scope of the present
technology, as defined by the appended claims. The examples can
include or incorporate any of the variations, aspects or
embodiments of the present technology described above. The
variations, aspects or embodiments described above may also further
each include or incorporate the variations of any or all other
variations, aspects or embodiments of the present technology.
Examples
[0132] Section 1.1
[0133] Materials and Instrumentation.
[0134] All solvents and reagents, unless otherwise noted, were
purchased from commercial sources and used as received without
further purification. Solvents noted as "dry" were obtained
following storage over 3 .ANG. molecular sieves. Reactions were
monitored by thin-layer chromatography (TLC, Whatman UV254
aluminum-backed silica gel). The HPLC system used for analysis and
purification of compounds consisted of a CBM-20A communications bus
module, an LC-20AP (preparative) pump, and an SPD-20AV UV/Vis
detector monitoring at 270 nm (Shimadzu, Japan). Purification was
performed with an Epic Polar preparative column, 120 .ANG., 10
.mu.m, 25 cm.times.20 mm (ES Industries, West Berlin, N.J.) at a
flow rate of 14 mL/min, unless otherwise noted. Gradient HPLC
methods were employed using a binary mobile phase that contained
H.sub.2O (A) and either MeOH (B) or ACN (C). HPLC Method A: 10% B
(0-5 min), 10-100% B (5-25 min). Method B: 10% C (0-5 min), 10-100%
C (5-25 min). Method C: 10% C (0-5 min), 10-100% C (5-40 min).
Method D: 10% C (0-5 min), 10-100% C (5-20 min). The solvent
systems contained 0.2% trifluoroacetic acid (TFA). NMR spectra were
recorded at ambient temperature on Varian Inova 300 MHz, 400 MHz,
500 MHz or 600 MHz spectrometers, or on a Bruker AV III HD 500 MHz
spectrometer equipped with a broadband Prodigy cryoprobe. Chemical
shifts are reported in ppm. .sup.1H and .sup.13C NMR spectra were
referenced to the TMS internal standard (0 ppm), to the residual
solvent peak, or to an acetonitrile internal standard (2.06 ppm in
D.sub.2O spectra). .sup.19F NMR spectra were referenced to a
monofluorobenzene internal standard (-113.15 ppm). The splitting of
proton resonances in the reported .sup.1H spectra is defined as:
s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, dt=doublet
of triplets, td=triplet of doublets, and br=broad. IR spectroscopy
was performed on a KBr pellet of sample using a Nicolet Avatar 370
DTGS (ThermoFisher Scientific, Waltham, Mass.). High-resolution
mass spectra (IRMS) were recorded on an Exactive Orbitrap mass
spectrometer in positive ESI mode (ThermoFisher Scientific,
Waltham, Mass.). UV/visible spectra were recorded on a Cary 8454
UV-Vis (Agilent Technologies, Santa Clara, Calif.) using 1-cm
quartz cuvettes, unless otherwise noted. Elemental analysis (EA)
was performed by Atlantic Microlab, Inc. (Norcross, Ga.).
Preparation of di-tert-butyl
((1-(tert-butoxy)-6-(3-(3-ethynylphenyl)ureido)-1-oxohexan-2-yl)carbamoyl-
)glutamate (5)
##STR00007##
[0135] (S)-2-[(Imidazole-1-carbonyl)amino]pentanedioic Acid
di-tertbutyl Ester (2)
[0136] To a suspension of L-di-tert-butyl glutamate hydrochloride
(15.0 g, 51 mmol) in DCM (150 mL) cooled to 0.degree. C. was added
TEA (18 mL) and DMAP (250 mg). After the mixture was stirred for 5
min, CDI (9.0 g, 56 mmol) was added and the mixture was stirred
overnight with warming to room temperature. The mixture was diluted
with DCM (150 mL) and washed with saturated sodium bicarbonate (60
mL), water (2.times.100 mL), and brine (100 mL). The organic layer
was dried over sodium sulfate and concentrated to afford the crude
product as a semi-solid, which slowly solidified upon standing. The
crude material was triturated with hexane/ethyl acetate to afford a
white solid which was filtered, washed with hexane (100 mL), and
dried to afford 2 (15.9 g, 45 mmol, 88%) as a white solid.
(S)-2-[3((S)-(5-Benzyloxycarbonylamino)-1-tert-butoxycarbonylpentylureido]-
pentanedioic Acid di-tert-butyl Ester (3)
[0137] To a solution of 2 (1 g, 2.82 mmol) in DCE (10 mL) at
0.degree. C. was added MeOTf (0.47 g, 2.85 mmol) and TEA (0.57 g,
5.65 mmol). After the solution was stirred for 30 min,
Cbz-L-Lys-Ot-Bu (1.06 g, 2.82 mmol) was added in one portion and
allowed to stir for 1 h at 40.degree. C. The mixture was
concentrated to dryness and purified by column chromatography
(SiO.sub.2) to afford 3 as a white solid (1.37 g, 79%).
2-[3-(5-Amino-1-tert-butoxycarbonylpentyl)ureido]pentanedioic Acid
di-tert-butyl ester (4)
[0138] To a solution of 3 (630 mg, 1.0 mmol) in ethanol (20 mL)
under a hydrogen atmosphere was added ammonium formate (630 mg, 10
eq) followed by 10% Pd--C. The suspension was allowed to stand with
occasional agitation overnight until complete. The mixture was
filtered through Celite and concentrated to afford the desired
product (479 mg, 98%) as a waxy solid.
Di-tert-butyl
((1-(tert-butoxy)-6-(3-(3-ethynylphenyl)ureido)-1-oxohexan-2-yl)carbamoyl-
)glutamate (5)
[0139] To a solution of 4 (0.488, 1 mmol) in DCM (10 mL) was added
1-ethynyl-3-isocyanatobenzene (185 mg, 1.3 mmol) in DCM (5 mL) at
r.t under N.sub.2. The resulting reaction mixture was stirred for
12 h at the same temperature and transferred to a separating funnel
and washed with water (2.times.50 mL) and brine (30 mL). The
organic layer was dried over sodium sulfate and concentrated to
afford the crude product as a semisolid which was purified by
column chromatography (SiO.sub.2) to afford 5 as a white foam
(84%).
Preparation of
tert-butyl-N2-(N2-(((9H-fluoren-9-yl)methoxy)carbonyl)
glycylglycylglycyl-N6-(tert-butoxycarbonyl)lysyl)-N6-((benzyloxy)carbonyl-
)-L-lysinate (10)
##STR00008##
[0140] 2,5-dioxopyrrolidin-1-yl
N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(tert-butoxycarbonyl)-L-lysina-
te (7)
[0141]
N2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N6-(tert-butoxycarbonyl)-L--
lysine 6 (4.68 g, 10 mmol) was dissolved in dry DCM (20 mL) and
DIPEA (1.74 mL, 10 mmol) was added. The reaction mixture was
stirred at r.t. for 10 min and solid di(N-succinimidyl) carbonate
(3.84 g, 15 mmol) was added in one portion. The resulting reaction
mixture was stirred for 3-4 h and diluted with DCM, transferred to
a separating funnel and washed with an excess of water. The organic
layer was collected, dried on MgSO.sub.4 and evaporated to dryness
to afford a semi-solid. The crude product was recrystallized from
ethanol and diethyl ether to give 7 as a cream colored solid (3.44
g, 61%).
tert-butyl
N2-(N2-(((9H-fluoren-9-yl)oxy)carbonyl)-N6-(tert-butoxycarbonyl-
)-L-lysyl)-N6-((benzyloxy)carbonyl)-L-lysinate (8)
[0142] To a suspension of H-Lys(Z)-OtBu HCl (3.72 g, 10 mmol) in
DCM (25 mL) was added DIPEA (1.74 mL, 10 mmol) at 0.degree. C.
followed by dropwise addition of compound 7 (5.65 g, 10 mmol) in
DCM (20 mL). The resulting clear solution was stirred overnight at
r.t. The solvent was evaporated and the crude compound was purified
by column chromatography (SiO.sub.2) to afford 8 as a white solid
(76%).
tert-butyl
N6-((benzyloxy)carbonyl)-N2-(N6-(tert-butoxycarbonyl)-L-lysyl)--
L-lysinate (9)
[0143] To a solution of compound 8 (1.156 g, 2 mmol) in DCM was
added diethylamine (6 mL) dropwise and the resulting reaction
mixture was stirred at r.t for 4-5 h. Solvents were evaporated
under reduced pressure and the crude product was re-dissolved in
DCM and washed with water (2.times.100 mL), and brine (100 mL). The
organic layer was dried over sodium sulfate and concentrated to
afford the crude product as a semisolid, which was used as such
without any further purification.
tert-butyl-N2-(N2-(((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycylglycyl-N-
6-(tert-butoxycarbonyl)-L-lysyl)-N6-((benzyloxy)carbonyl)-L-lysinate
(10)
[0144] To a solid mixture of
(((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycylglycine (246 mg,
0.6 mmol) and HATU (230 mg, 0.6 mmol) under N.sub.2 was added dry
DMF, and the mixture was stirred for 5 min at r.t. DIPEA (0.12 mL,
0.7 mmol) was added to the reaction mixture and stirring was
continued for 10 min at r.t. A solution of compound 9 (282 mg, 0.5
mmol) in DMF was added dropwise at r.t and stirred at the same
temperature for 12 h. DMF was evaporated under reduced pressure to
give a suspension, which was dissolved in DCM (10 mL), transferred
to a separating funnel and washed with water (2.times.20 mL) and
brine (15 mL). The organic layer was collected, dried on MgSO.sub.4
and evaporated to dryness to afford a semi-solid. The crude
compound was purified by column chromatography (SiO.sub.2) to
afford the desired product 10 as a brown solid (41%).
tert-butyl
N2-(N2-(2-azidoacetyl)glycylglycylglycyl-N6-(tert-butoxycarbony- l)
lysyl)-N6-((benzyloxy)carbonyl)-L-lysinate (12)
##STR00009##
[0145] tert-butyl
N6-((benzyloxy)carbonyl)-N2-(N6-(tert-butoxycarbonyl)-N2-glycylglycylglyc-
yl-L-lysyl)-L-lysinate (11)
[0146] To a solution of compound 10 (0.478 g, 0.5 mmol) in DCM (10
mL) was added diethylamine (2 mL) dropwise and the resulting
reaction mixture was stirred at r.t for 3 h. Solvents were
evaporated under reduced pressure and re-dissolved in DCM and
washed with water (2.times.20 mL) and brine (20 mL). The organic
layer was dried over sodium sulfate and concentrated to afford the
crude product 11 as a semisolid, which was used without any further
purification.
tert-butyl
N2-(N2-(2-azidoacetyl)glycylglycylglycyl-N6-(tert-butoxycarbony-
l)-L-lysyl)-N6-((benzyloxy)carbonyl)-L-lysinate (12)
[0147] To a solid mixture of azidoacetic acid (101 mg, 1 mmol) and
HATU (383 mg, 1 mmol) under N.sub.2 was added dry DMF (5 mL), and
the mixture was stirred for 5 min at r.t. DIPEA (0.17 mL, 1 mmol)
was added to the reaction mixture and stirring was continued for 10
min at r.t. A solution of compound 11 (367 mg, 0.5 mmol) in DMF (5
mL) was added dropwise at r.t and stirred at the same temperature
for 12 h. DMF was evaporated under reduced pressure to give a
suspension, which was dissolved in DCM (10 mL) and washed with
water (2.times.20 mL), and brine (15 mL). The crude compound was
used without any further purification.
Preparation of
10-(6-alkamido-1-(tert-butoxy)-1-oxohexan-2-yl)-24,28,30-tri-tert-butyl-2-
,2-dimethyl-4,12,21,26-tetraoxo-3-oxa-5,11,20,25,27-pentaazatriacontane-10-
,24,28,30-tetracarboxylate (14)
##STR00010##
[0148]
di-tert-butyl(((S)-1-(tert-butoxy)-6-(3-(3-(1-((9S,12S)-9-(tert-but-
oxycarbonyl)-12-(4-((tert-butoxycarbonyl)amino)butyl)-3,11,14,17,20,23-hex-
aoxo-1-phenyl-2-oxa-4,10,13,16,19,22-hexaazatetracosan-24-yl)-1H-1,2,3-tri-
azol-5-yl)phenyl)ureido)-1-oxohexan-2-yl)carbamoyl)-L-glutamate
(13)
[0149] Compound 12 (140 mg, 0.1 mmol) and compound 5 (63 mg, 0.1
mmol) were dissolved in DMF (2 mL) and aqueous solutions of 0.5M
CuSO.sub.4 and 0.5M sodium ascorbate were added subsequently. The
resulting reaction was stirred for 3 h at r.t. DMF was evaporated
and the crude compound 13 was used without any further
purification.
10-(6-alkamido-1-(tert-butoxy)-1-oxohexan-2-yl)-24,28,30-tri-tert-butyl-2,-
2-dimethyl-4,12,21,26-tetraoxo-3-oxa-5,11,20,25,27-pentaazatriacontane-10,-
24,28,30-tetracarboxylate (14)
[0150] Compound 13 (144 mg, 0.1 mmol) was dissolved in a mixture of
methanol.THF (1:1, 10 mL) and 10% Pd--C was added. The resulting
suspension was stirred under H.sub.2 (balloon pressure) atmosphere
for 3 h. The mixture was filtered through Celite and concentrated
to afford the corresponding amine (not shown) as semi solid, which
was used immediately to the next step. To a solid mixture of acid
RCOOH (0.1 mmol) and HATU (38 mg, 0.1 mmol) under N.sub.2 was added
dry DMF (3 mL) and the mixture was stirred for at r.t. for 5 min.
DIPEA (0.017 mL, 0.1 mmol) was added to the reaction mixture and
stirring was continued for 10 min at r.t. A solution of amine (0.1
mmol) in DMF (2 mL) was added dropwise at r.t and stirred at the
same temperature for 12 h. DMF was evaporated under reduced
pressure to give a suspension, which was dissolved in DCM (5 mL)
and washed with water (2.times.10 mL) and brine (10 mL). The
organic layer was dried over sodium sulfate and concentrated to
afford the crude product which was purified by column
chromatography (SiO.sub.2), and product 14 was isolated as a
semi-solid.
Preparation of 15
##STR00011##
[0152] To a solution of compound 14 (1 eq;
R=(4-iodophenyl)CH.sub.2--) in dioxane (2 mL) was added 4M HCl in
dioxane (2 mL). The resulting reaction mixture was stirred for 3 h
at r.t. Completion of the reaction was monitored by TLC. Solvents
were removed under reduced pressure and co-distilled with toluene
(2.times.5 mL). The amine HCl salts formed were dissolved in DMF
(1.5 mL) and DIPEA (0.5 mmol, 20 eq) was added. The resulting
reaction mixture was stirred for 10 min before adding p-NCS-Bn-DOTA
(2 eq) and distilled water (0.5 mL). Stirring was continued for 3 h
at r.t. The reaction mixture was directly subjected to LCMS
purification using 0.1% formic acid in ACN and water. The product
was collected and lyophilized. .sup.1H NMR (500 MHz, DMSO-d.sub.6):
.delta. 12.25 (bs, 7H), 9.40 (bs, 1H), 8.64-8.62 (m, 1H, N--H),
8.54-8.52 (m, 1H, N--H), 8.42 (s, 1H), 8.34-8.31 (m, 1H, N--H),
8.15-8.11 (m, 3H), 8.04-8.02 (m, 1H, N--H), 7.94-7.91 (m, 2H,
N--H), 7.64-7.63 (m, 3H), 7.37-7.31 (m, 5H), 7.28-7.25 (m, 1H),
7.17-7.16 (m, 2H), 7.05-7.04 (m, 3H), 6.34-6.30 (m, 2H), 6.17 (bs,
1H), 5.21 (s, 2H), 4.34-4.30 (m, 1H), 4.13-4.04 (m, 4H), 3.84-3.83
(m, 2H), 3.75-3.74 (m, 4H), 3.63-3.60 (m, 3H), 3.16-3.13 (m, 4H),
3.12-3.05 (m, 4H), 3.02-2.98 (m, 6H), 2.30-2.18 (m, 3H), 1.95-1.88
(m, 1H), 1.71-1.65 (m, 5H), 1.58-1.49 (m, 6H), 1.45-1.22 (m, 12H).
.sup.13C NMR (500 MHz, DMSO-d.sub.6): .delta. 174.3, 174.0, 173.5,
173.2, 171.4, 169.3, 168.9, 168.7, 168.2, 165.6, 157.1, 154.9,
146.1, 140.9, 136.7, 136.1, 131.2, 130.8, 129.0, 122.6, 118.4,
117.8, 116.9, 116.0, 113.9, 53.4, 52.1, 51.9, 51.6, 51.4, 41.9,
41.8, 41.6, 41.5, 38.2, 31.8, 31.7, 30.3, 29.7, 29.3, 28.5, 28.0,
27.3, 22.7, 22.5, 22.4, 17.9, 16.5, 12.3. HRMS calculated for
C.sub.73H.sub.102IN.sub.19O.sub.24S ([M+2H].sup.+), 1787.6110,
found 1787.6048.
Preparation of
2-[3-(5-Amino-1-tert-butoxycarbonylpentyl)ureido]pentanedioic Acid
di-tert-butyl Ester (17)
##STR00012##
[0153] 5-benzyl 1-(tert-butyl)
(((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)carbamoyl)-L-glutamate
(16)
[0154] To a solution of 2 (1 g, 2.82 mmol) in DCE (10 mL) at
0.degree. C. was added MeOTf (0.47 g, 2.85 mmol) and TEA (0.57 g,
5.65 mmol). After the solution was stirred for 30 min,
H-L-Glu(Bzl)-OtBu hydrochloride (0.927 g, 2.82 mmol) was added in
one portion and allowed to stir for 1 h at 40.degree. C. The
mixture was concentrated to dryness and purified by column
chromatography (SiO.sub.2) to afford the desired product as a white
solid (79%).
(S)-5-(tert-butoxy)-4-(3-((S)-1,5-di-tert-butoxy-1,5-dioxopentan-2-yl)urei-
do)-5-oxopentanoic Acid (17)
[0155] To a solution of 16 in ethanol (20 mL) under a hydrogen
atmosphere was added ammonium formate (630 mg, 10 eqv) followed by
10% Pd--C, and the suspension was allowed to stand with occasional
agitation overnight until complete. The mixture was filtered
through Celite and concentrated to afford 17, the desired product
(479 mg, 98%) as a waxy solid.
Preparation of 2,5-dioxopyrrolidin-1-yl
8-((((9H-fluoren-9-yl)methoxy)carbonyl) amino)octanoate (19)
##STR00013##
[0157] 8-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)octanoic acid,
18 (1.43 g, 3 mmol) was dissolved in anhydrous DCM (20 mL) and
DIPEA (0.522 mL, 3 mmol) was added. The reaction mixture was
stirred at r.t for 10 min and solid di(N-succinimidyl) carbonate
(1.152 g, 4.5 mmol) was added in one portion. The resulting
reaction mixture was stirred for 3 h and diluted with DCM and
transferred in to a separating funnel and washed with excess of
water. The organic layer was collected, dried on MgSO.sub.4 and
evaporated to dryness to afford a semi-solid, which was
recrystallized from ethanol and diethyl ether to give the desired
product as an off-white solid (0.932 g, 65.08%).
Preparation of tetra-tert-butyl
(3S,7S,21S,24S)-28-amino-21-(4-((tert-butoxycarbonyl)amino)butyl)-5,10,19-
,22-tetraoxo-4,6,11,20,23-pentaazaoctacosane-1,3,7,24-tetracarboxylate
(23)
##STR00014##
[0158] tert-butyl
N2-(N2-(8-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)octanoyl)-N6-(tert-b-
utoxycarbonyl)-L-lysyl)-N6-((benzyloxy)carbonyl)-L-lysinate
(20)
[0159] Compound 9 (0.551 g, mmol) and compound 19 (0.573, 1.2 mmol)
were dissolved in DCM (10 mL) and stirred for 12 h at r.t. Progress
of the reaction was monitored by TLC. The mixture was concentrated
to dryness and purified by column chromatography (SiO.sub.2) to
afford the desired product 20 as a brown solid (41%).
tert-butyl
N2-(N2-(8-aminooctanoyl)-N6-(tert-butoxycarbonyl)-L-lysyl)-N6-(-
(benzyloxy)carbonyl)-L-lysinate (21)
[0160] To a solution of compound 20 (0.457 g, 0.5 mmol) in DCM (10
mL) was added diethylamine (3 mL) dropwise and the resulting
reaction mixture was stirred at r.t for 3 h. Solvents were
evaporated under reduced pressure and re-dissolved in DCM and
washed with water (2.times.20 mL) and brine (20 mL). The organic
layer was dried over sodium sulfate and concentrated to afford the
crude product 21 as a semisolid, which was used without any further
purification.
tetra-tert-butyl
(9S,12S,26S,30S)-12-(4-((tert-butoxycarbonyl)amino)butyl)-3,11,14,23,28-p-
entaoxo-1-phenyl-2-oxa-4,10,13,22,27,29-hexaazadotriacontane-9,26,30,32-te-
tracarboxylate (22)
[0161] To a solid mixture of compound 17 (0.114, 0.24 mmol) and
HATU (0.092 g, 0.24 mmol) under N.sub.2 was added dry DMF, and the
mixture was stirred for 5 min at r.t. DIPEA (0.041 mL, 0.24 mmol)
was added to the reaction mixture and stirring was continued for 10
min at r.t. A solution of compound 21 (0.141, 0.2 mmol) in DMF was
added dropwise at r.t and stirred at the same temperature for 12 h.
DMF was evaporated under reduced pressure to give a suspension,
which was dissolved in DCM (10 mL) and washed with water
(2.times.20 mL), and brine (15 mL). The organic layer was dried
over sodium sulfate and concentrated to afford the crude product
which was purified by column chromatography (SiO.sub.2) to afford
the product 22 as a semi-solid (28%).
tetra-tert-butyl
(3S,7S,21S,24S)-28-amino-21-(4-((tert-butoxycarbonyl)amino)butyl)-5,10,19-
,22-tetraoxo-4,6,11,20,23-pentaazaoctacosane-1,3,7,24-tetracarboxylate
(23)
[0162] Compound 22 (0.1 g, 0.085 mmol) was dissolved in a mixture
of methanol.THF (1:1, 10 mL) and 10% Pd--C was added. The resulting
suspension was stirred under H.sub.2 (balloon pressure) atmosphere
for 3 h. The mixture was filtered through Celite and concentrated
to afford the desired product 23 (91%) as a waxy solid.
[0163] Preparation of 24 (a-2) and 25 (a-h)
TABLE-US-00001 ##STR00015## 24 (a-g) or 22 ##STR00016## 25 (a-h)
Com- pound 24a & 25a 24h & 25b 24c & 25c 24d & 25d
24e & 25e 24f & 25f 24g & 25g 22 & 25h n = 3 3 3 3
2 1 0 0 R = ##STR00017## ##STR00018## ##STR00019## ##STR00020##
##STR00021## ##STR00022## ##STR00023## ##STR00024##
##STR00025##
tetra-tert-butyl
(2S)-5-(4-((tert-butoxycarbonyl)amino)butyl)-2-(4-(4-(4-iodophenyl)butana-
mido)butyl)-1,4,7,16,21-pentaoxo-3,6,15,20,22-pentaazapentacosane-1,19,23,-
25-tetracarboxylate (24a)
[0164] To a solid mixture of 4-(4-iodophenyl)butanoic acid (0.029
g, 0.1 mmol) and HATU (0.038 g, 0.1 mmol) under N.sub.2 was added
dry DMF (2 mL) and the mixture was stirred for at r.t. for 5 min.
DIPEA (0.017 mL, 0.1 mmol) was added to the reaction mixture and
stirring was continued for 10 min at r.t. A solution of compound 23
(0.052, 0.05 mmol) in DMF (1 mL) was added dropwise at r.t and
stirred at the same temperature for 12 h. DMF was evaporated under
reduced pressure to give a suspension, which was dissolved in DCM
(5 mL) and washed with water (2.times.10 mL) and brine (10 mL). The
organic layer was dried over sodium sulfate and concentrated to
afford the crude product which was purified by column
chromatography (SiO.sub.2), and product 24a was isolated as a
semi-solid (18%).
##STR00026##
33-(4-iodophenyl)-5,10,19,22,30-pentaoxo-21-(4-(3-(4-((1,4,7,10-tetrakis(-
carboxymethyl)-1,4,7,10-tetraazacyclododecan-2-yl)methyl)phenyl)thioureido-
)butyl)-4,6,11,20,23,29-hexaazatritriacontane-1,3,7,24-tetracarboxylic
Acid (25a)
[0165] definted, but the a solution of compound 24a (0.025 mmol, 1
eq) in dioxane (2 mL) was added 4M HCl in dioxane (2 mL). The
resulting reaction mixture was stirred for 3 h at r.t. Completion
of the reaction was monitored by TLC. Solvents were removed under
reduced pressure and co-distilled with toluene (2.times.5 mL). The
amine HCl salts were dissolved in DMF (1.5 mL) and DIPEA (0.5 mmol,
20 eq) was added. The resulting reaction mixture was stirred for 10
min before adding p-NCS-Bn-DOTA (0.050 mmol, 2eq) and distilled
water (0.5 mL). Stirring was continued for 3 h at r.t. The reaction
mixture was directly subjected to LCMS purification using 0.1%
formic acid in ACN and water.
##STR00027##
tetra-tert-butyl
(2S)-2-(4-(4-(4-bromophenyl)butanamido)butyl)-5-(4-((tert-butoxycarbonyl)-
amino)butyl)-1,4,7,16,21-pentaoxo-3,6,15,20,22-pentaazapentacosane-1,19,23-
,25-tetracarboxylate (24b)
[0166] To a solid mixture of 4-(4-bromophenyl)butanoic acid (0.024
g, 0.1 mmol) and HATU (0.038 g, 0.1 mmol) under N.sub.2 was added
dry DMF (2 mL) and the mixture was stirred for at r.t. for 5 min.
DIPEA (0.017 mL, 0.1 mmol) was added to the reaction mixture and
stirring was continued for 10 min at r.t. A solution of compound 23
(0.052, 0.05 mmol) in DMF (1 mL) was added dropwise at r.t and
stirred at the same temperature for 12 h. DMF was evaporated under
reduced pressure to give a suspension, which was dissolved in DCM
(5 mL) and washed with water (2.times.10 mL), and brine (10 mL).
The organic layer was dried over sodium sulfate and concentrated to
afford the crude product which was purified by column
chromatography (SiO.sub.2), and product 24b was isolated as a
semi-solid (6%).
##STR00028##
33-(4-bromophenyl)-5,10,19,22,30-pentaoxo-21-(4-(3-(4-((1,4,7,10-tetrakis-
(carboxymethyl)-1,4,7,10-tetraazacyclododecan-2-yl)methyl)phenyl)thioureid-
o)butyl)-4,6,11,20,23,29-hexaazatritriacontane-1,3,7,24-tetracarboxylic
Acid (25b)
[0167] To a solution of compound 24b (0.020 mmol, 1 eq) in dioxane
(2 mL) was added 4M HCl in dioxane (2 mL). The resulting reaction
mixture was stirred for 3 h at r.t. Completion of the reaction was
monitored by TLC. Solvents were removed under reduced pressure and
co-distilled with toluene (2.times.5 mL). The amine HCl salts were
dissolved in DMF (1.5 mL) and DIPEA (0.5 mmol, 20 eq) was added.
The resulting reaction mixture was stirred for 10 min before adding
p-NCS-Bn-DOTA (0.050 mmol, 2eq) and distilled water (0.5 mL).
Stirring was continued for 3 h at r.t. The reaction mixture was
directly subjected to LCMS purification using 0.1% formic acid in
ACN and water to afford 25b.
##STR00029##
tetra-tert-butyl(2S,5S,19S,23S)-5-(4-((tert-butoxycarbonyl)amino)butyl)-2-
-(4-(4-(4-iodophenyl)butanamido)butyl)-1,4,7,16,21-pentaoxo-3,6,15,20,22-p-
entaazapentacosane-1,19,23,25-tetracarboxylate (24a)
[0168] To a solid mixture of 4-(4-iodophenyl)butanoic acid (0.029
g, 0.1 mmol) and HATU (0.038 g, 0.1 mmol) under N.sub.2 was added
dry DMF (2 mL) and the mixture was stirred for at r.t. for 5 min.
DIPEA (0.017 mL, 0.1 mmol) was added to the reaction mixture and
stirring was continued for 10 min at r.t. A solution of compound 23
(0.052, 0.05 mmol) in DMF (1 mL) was added dropwise at r.t and
stirred at the same temperature for 12 h. DMF was evaporated under
reduced pressure to give a suspension, which was dissolved in DCM
(5 mL) and washed with water (2.times.10 mL) and brine (10 mL). The
organic layer was dried over sodium sulfate and concentrated to
afford the crude product which was purified by column
chromatography (SiO.sub.2), and product 24a was isolated as a
semi-solid (18%).
##STR00030##
(3S,7S,21S,24S)-33-(4-iodophenyl)-5,10,19,22,30-pentaoxo-21-(4-(3-(4-((1,-
4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecan-2-yl)methyl)p-
henyl)thioureido)butyl)-4,6,11,20,23,29-hexaazatritriacontane-1,3,7,24-tet-
racarboxylic Acid (25a)
[0169] definted, but the a solution of compound 24a (0.025 mmol, 1
eq) in dioxane (2 mL) was added 4M HCl in dioxane (2 mL). The
resulting reaction mixture was stirred for 3 h at r.t. Completion
of the reaction was monitored by TLC. Solvents were removed under
reduced pressure and co-distilled with toluene (2.times.5 mL). The
amine HCl salts were dissolved in DMF (1.5 mL) and DIPEA (0.5 mmol,
20 eq) was added. The resulting reaction mixture was stirred for 10
min before adding p-NCS-Bn-DOTA (0.050 mmol, 2eq) and distilled
water (0.5 mL). Stirring was continued for 3 h at r.t. The reaction
mixture was directly subjected to LCMS purification using 0.1%
formic acid in ACN and water.
##STR00031##
tetra-tert-butyl
(2S,5S,19S,23S)-2-(4-(4-(4-bromophenyl)butanamido)butyl)-5-(4-((tert-buto-
xycarbonyl)amino)butyl)-1,4,7,16,21-pentaoxo-3,6,15,20,22-pentaazapentacos-
ane-1,19,23,25-tetracarboxylate (24b)
[0170] To a solid mixture of 4-(4-bromophenyl)butanoic acid (0.024
g, 0.1 mmol) and HATU (0.038 g, 0.1 mmol) under N.sub.2 was added
dry DMF (2 mL) and the mixture was stirred for at r.t. for 5 min.
DIPEA (0.017 mL, 0.1 mmol) was added to the reaction mixture and
stirring was continued for 10 min at r.t. A solution of compound 23
(0.052, 0.05 mmol) in DMF (1 mL) was added dropwise at r.t and
stirred at the same temperature for 12 h. DMF was evaporated under
reduced pressure to give a suspension, which was dissolved in DCM
(5 mL) and washed with water (2.times.10 mL), and brine (10 mL).
The organic layer was dried over sodium sulfate and concentrated to
afford the crude product which was purified by column
chromatography (SiO.sub.2), and product 24b was isolated as a
semi-solid (6%).
##STR00032##
(3S,7S,21S,24S)-33-(4-bromophenyl)-5,10,19,22,30-pentaoxo-21-(4-(3-(4-((1-
,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecan-2-yl)methyl)-
phenyl)thioureido)
butyl)-4,6,11,20,23,29-hexaazatritriacontane-1,3,7,24-tetracarboxylic
Acid (25b)
[0171] To a solution of compound 24b (0.020 mmol, 1 eq) in dioxane
(2 mL) was added 4M HCl in dioxane (2 mL). The resulting reaction
mixture was stirred for 3 h at r.t. Completion of the reaction was
monitored by TLC. Solvents were removed under reduced pressure and
co-distilled with toluene (2.times.5 mL). The amine HCl salts were
dissolved in DMF (1.5 mL) and DIPEA (0.5 mmol, 20 eq) was added.
The resulting reaction mixture was stirred for 10 min before adding
p-NCS-Bn-DOTA (0.050 mmol, 2eq) and distilled water (0.5 mL).
Stirring was continued for 3 h at r.t. The reaction mixture was
directly subjected to LCMS purification using 0.1% formic acid in
ACN and water to afford 25b.
##STR00033##
tetra-tert-butyl
(2S,5S,19S,23S)-5-(4-((tert-butoxycarbonyl)amino)butyl)-1,4,7,16,21-penta-
oxo-2-(4-(4-(p-tolyl)butanamido)butyl)-3,6,15,20,22-pentaazapentacosane-1,-
19,23,25-tetracarboxylate (24c)
[0172] To a solid mixture of 4-(p-tolyl)butanoic acid (0.0178 g,
0.1 mmol) and HATU (0.038 g, 0.1 mmol) under N.sub.2 was added dry
DMF (2 mL) and the mixture was stirred for at r.t. for 5 min. DIPEA
(0.017 mL, 0.1 mmol) was added to the reaction mixture and stirring
was continued for 10 min at r.t. A solution of compound 23 (0.052,
0.05 mmol) in DMF (1 mL) was added dropwise at r.t and stirred at
the same temperature for 12 h. DMF was evaporated under reduced
pressure to give a suspension, which was dissolved in DCM (5 mL)
and washed with water (2.times.10 mL), and brine (10 mL). The
organic layer was dried over sodium sulfate and concentrated to
afford the crude product which was purified by column
chromatography (SiO.sub.2), and product 24c was isolated as a
semi-solid (18%).
##STR00034##
(3S,7S,21S,24S)-5,10,19,22,30-pentaoxo-21-(4-(3-(4-((1,4,7,10-tetrakis(ca-
rboxymethyl)-1,4,7,10-tetraazacyclododecan-2-yl)methyl)phenyl)thioureido)b-
utyl)-33-(p-tolyl)-4,6,11,20,23,29-hexaazatritriacontane-1,3,7,24-tetracar-
boxylic Acid (25c)
[0173] To a solution of compound 24c (0.03 mmol, 1 eq) in dioxane
(2 mL) was added 4M HCl in dioxane (2 mL). The resulting reaction
mixture was stirred for 3 h at r.t. Completion of the reaction was
monitored by TLC. Solvents were removed under reduced pressure and
co-distilled with toluene (2.times.5 mL). The amine HCl salts were
dissolved in DMF (1.5 mL) and DIPEA (0.5 mmol, 20 eq) was added.
The resulting reaction mixture was stirred for 10 min before adding
p-NCS-Bn-DOTA (0.050 mmol, 2eq) and distilled water (0.5 mL).
Stirring was continued for 3 h at r.t. The reaction mixture was
directly subjected to LCMS purification using 0.1% formic acid in
ACN and water.
##STR00035##
tetra-tert-butyl
(2S,5S,19S,23S)-5-(4-((tert-butoxycarbonyl)amino)butyl)-1,4,7,16,21-penta-
oxo-2-(4-(4-phenylbutanamido)butyl)-3,6,15,20,22-pentaazapentacosane-1,19,-
23,25-tetracarboxylate (24d)
[0174] To a solid mixture of 4-phenylbutanoic acid (0.0164 g, 0.1
mmol) and HATU (0.038 g, 0.1 mmol) under N.sub.2 was added dry DMF
(2 mL) and the mixture was stirred for at r.t. for 5 min. DIPEA
(0.017 mL, 0.1 mmol) was added to the reaction mixture and stirring
was continued for 10 min at r.t. A solution of compound 23 (0.052,
0.05 mmol) in DMF (1 mL) was added dropwise at r.t and stirred at
the same temperature for 12 h. DMF was evaporated under reduced
pressure to give a suspension, which was dissolved in DCM (5 mL)
and washed with water (2.times.10 mL), and brine (10 mL). The
organic layer was dried over sodium sulfate and concentrated to
afford the crude product which was purified by column
chromatography (SiO.sub.2), and product 24d was isolated as a
semi-solid (21%).
##STR00036##
(3S,7S,21S,24S)-5,10,19,22,30-pentaoxo-33-phenyl-21-(4-(3-(4-((1,4,7,10-t-
etrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecan-2-yl)methyl)phenyl)th-
ioureido)butyl)-4,6,11,20,23,29-hexaazatritriacontane-1,3,7,24-tetracarbox-
ylic Acid (25d)
[0175] To a solution of compound 24d (0.04 mmol, 1 eq) in dioxane
(3 mL) was added 4M HCl in dioxane (3 mL). The resulting reaction
mixture was stirred for 3 h at r.t. Completion of the reaction was
monitored by TLC. Solvents were removed under reduced pressure and
co-distilled with toluene (2.times.5 mL). The amine HCl salts were
dissolved in DMF (1.5 mL) and DIPEA (0.5 mmol, 20 eq) was added.
The resulting reaction mixture was stirred for 10 min before adding
p-NCS-Bn-DOTA (0.080 mmol, 2eq) and distilled water (0.5 mL).
Stirring was continued for 3 h at r.t. The reaction mixture was
directly subjected to LCMS purification using 0.1% formic acid in
ACN and water to afford 25d.
##STR00037##
tetra-tert-butyl
(2S,5S,19S,23S)-5-(4-((tert-butoxycarbonyl)amino)butyl)-1,4,7,16,21-penta-
oxo-2-(4-(4-phenylbutanamido)butyl)-3,6,15,20,22-pentaazapentacosane-1,19,-
23,25-tetracarboxylate (24e)
[0176] To a solid mixture of
4-oxo-4-(5,6,7,8-tetrahydronaphthalen-2-yl)butanoic acid (0.023 g,
0.1 mmol) and HATU (0.038 g, 0.1 mmol) under N.sub.2 was added dry
DMF (2 mL) and the mixture was stirred for at r.t. for 5 min. DIPEA
(0.017 mL, 0.1 mmol) was added to the reaction mixture and stirring
was continued for 10 min at r.t. A solution of compound 23 (0.052,
0.05 mmol) in DMF (1 mL) was added dropwise at r.t and stirred at
the same temperature for 12 h. DMF was evaporated under reduced
pressure to give a suspension, which was dissolved in DCM (5 mL)
and washed with water (2.times.10 mL), and brine (10 mL). The
organic layer was dried over sodium sulfate and concentrated to
afford the crude product which was purified by column
chromatography (SiO.sub.2), and product 24e was isolated as a
semi-solid (17%).
##STR00038##
(3S,7S,21S,24S)-5,10,19,22,30,33-hexaoxo-33-(5,6,7,8-tetrahydronaphthalen-
-2-yl)-21-(4-(3-(4-((1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyc-
lododecan-2-yl)
methyl)phenyl)thioureido)butyl)-4,6,11,20,23,29-hexaazatritriacontane-1,3-
,7,24-tetracarboxylic Acid (25e)
[0177] To a solution of compound 24e (0.02 mmol, 1 eq) in dioxane
(3 mL) was added 4M HCl in dioxane (3 mL). The resulting reaction
mixture was stirred for 3 h at r.t. Completion of the reaction was
monitored by TLC. Solvents were removed under reduced pressure and
co-distilled with toluene (2.times.5 mL). The amine HCl salts were
dissolved in DMF (1.5 mL) and DIPEA (0.5 mmol, 20 eq) was added.
The resulting reaction mixture was stirred for 10 min before adding
p-NCS-Bn-DOTA (0.080 mmol, 2eq) and distilled water (0.5 mL).
Stirring was continued for 3 h at r.t. The reaction mixture was
directly subjected to LCMS purification using 0.1% formic acid in
ACN and water to afford 25e.
##STR00039##
tetra-tert-butyl
(2S,5S,19S,23S)-5-(4-((tert-butoxycarbonyl)amino)butyl)-2-(4-(2-(4-iodoph-
enyl)acetamido)butyl)-1,4,7,16,21-pentaoxo-3,6,15,20,22-pentaazapentacosan-
e-1,19,23,25-tetracarboxylate (24f)
[0178] To a solid mixture of 2-(4-Idophenyl)acetic acid (0.026 g,
0.1 mmol) and HATU (0.038 g, 0.1 mmol) under N.sub.2 was added dry
DMF (2 mL) and the mixture was stirred for at r.t. for 5 min. DIPEA
(0.017 mL, 0.1 mmol) was added to the reaction mixture and stirring
was continued for 10 min at r.t. A solution of compound 23 (0.052,
0.05 mmol) in DMF (1 mL) was added dropwise at r.t and stirred at
the same temperature for 12 h. DMF was evaporated under reduced
pressure to give a suspension, which was dissolved in DCM (5 mL)
and washed with water (2.times.10 mL), and brine (10 mL). The
organic layer was dried over sodium sulfate and concentrated to
afford the crude product which was purified by column
chromatography (SiO.sub.2), and product 24f was isolated as a
semi-solid (10%).
##STR00040##
(8S,11S,25S,29S)-1-(4-iodophenyl)-2,10,13,22,27-pentaoxo-11-(4-(3-(4-((1,-
4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecan-2-yl)methyl)p-
henyl)thioureido)butyl)-3,9,12,21,26,28-hexaazahentriacontane-8,25,29,31-t-
etracarboxylic Acid (25f)
[0179] To a solution of compound 24f (0.02 mmol, 1 eq) in dioxane
(3 mL) was added 4M HCl in dioxane (3 mL). The resulting reaction
mixture was stirred for 3 h at r.t. Completion of the reaction was
monitored by TLC. Solvents was removed under reduced pressure and
co-distilled with toluene (2.times.5 mL). The amine HCl salts were
dissolved in DMF (1.5 mL) and DIPEA (0.5 mmol, 20 eq) was added.
The resulting reaction mixture was stirred for 10 min before adding
p-NCS-Bn-DOTA (0.080 mmol, 2eq) and distilled water (0.5 mL).
Stirring was continued for 3 h at r.t. The reaction mixture was
directly subjected to LCMS purification using 0.1% formic acid in
ACN and water to afford 25f.
##STR00041##
tetra-tert-butyl
(2S,5S,19S,23S)-2-(4-(1H-indole-2-carboxamido)butyl)-5-(4-((tert-butoxyca-
rbonyl)amino)butyl)-1,4,7,16,21-pentaoxo-3,6,15,20,22-pentaazapentacosane--
1,19,23,25-tetracarboxylate (24 g)
[0180] To a solid mixture of indole-2-acetic acid (0.016 g, 0.1
mmol) and HATU (0.038 g, 0.1 mmol) under N.sub.2 was added dry DMF
(2 mL) and the mixture was stirred for at r.t. for 5 min. DIPEA
(0.017 mL, 0.1 mmol) was added to the reaction mixture and stirring
was continued for 10 min at r.t. A solution of compound 23 (0.052,
0.05 mmol) in DMF (1 mL) was added dropwise at r.t and stirred at
the same temperature for 12 h. DMF was evaporated under reduced
pressure to give a suspension, which was dissolved in DCM (5 mL)
and washed with water (2.times.10 mL), and brine (10 mL). The
organic layer was dried over sodium sulfate and concentrated to
afford the crude product which was purified by column
chromatography (SiO.sub.2), and product 24 g was isolated as a
semi-solid (15%).
##STR00042##
(7S,10S,24S,28S)-1-(1H-indol-2-yl)-1,9,12,21,26-pentaoxo-10-(4-(3-(4-((1,-
4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecan-2-yl)methyl)p-
henyl)thioureido)butyl)-2,8,11,20,25,27-hexaazatriacontane-7,24,28,30-tetr-
acarboxylic Acid (25 g)
[0181] To a solution of compound 24g (0.02 mmol, 1 eq) in dioxane
(3 mL) was added 4M HCl in dioxane (3 mL). The resulting reaction
mixture was stirred for 3 h at r.t. Completion of the reaction was
monitored by TLC. Solvents were removed under reduced pressure and
co-distilled with toluene (2.times.5 mL). The amine HCl salts were
dissolved in DMF (1.5 mL) and DIPEA (0.5 mmol, 20 eq) was added.
The resulting reaction mixture was stirred for 10 min before adding
p-NCS-Bn-DOTA (0.080 mmol, 2eq) and distilled water (0.5 mL).
Stirring was continued for 3 h at r.t. The reaction mixture was
directly subjected to LCMS purification using 0.1% formic acid in
ACN and water to afford 25 g.
##STR00043##
(9S,12S,26S,30S)-3,11,14,23,28-pentaoxo-1-phenyl-12-(4-(3-(4-((1,4,7,10-t-
etrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecan-2-yl)methyl)phenyl)th-
ioureido)butyl)-2-oxa-4,10,13,22,27,29-hexaazadotriacontane-9,26,30,32-tet-
racarboxylic Acid (25h)
[0182] To a solution of compound 22 (produced as defined herein)
(0.02 mmol, 1 eq) in dioxane (3 mL) was added 4M HCl in dioxane (3
mL). The resulting reaction mixture was stirred for 3 h at r.t.
Completion of the reaction was monitored by TLC. Solvents were
removed under reduced pressure and co-distilled with toluene
(2.times.5 mL). The amine HCl salts were dissolved in DMF (1.5 mL)
and DIPEA (0.5 mmol, 20 eq) was added. The resulting reaction
mixture was stirred for 10 min before adding p-NCS-Bn-DOTA (0.080
mmol, 2 eq) and distilled water (0.5 mL). Stirring was continued
for 3 h at r.t. The reaction mixture was directly subjected to LCMS
purification using 0.1% formic acid in ACN and water to afford
25h.
[0183] Section 1.2.
Preparation of dimethyl 4-aminopyridine-2,6-dicarboxylate (204)
##STR00044##
[0185] Dimethyl 4-azidopyridine-2,6-dicarboxylate.sup.245 (0.9445
g, 4.0 mmol), 10% Pd/C (0.1419 g), and DCM:MeOH (1:1, 18 mL) were
combined in a round-bottom flask. After purging the flask with a
balloon of H.sub.2, the reaction was stirred vigorously at room
temperature under an H.sub.2 atmosphere for 46 h. The gray mixture
was diluted with DMF (450 mL) and filtered through a bed of Celite.
Following a subsequent filtration through a 0.22 m nylon membrane,
the filtrate was concentrated at 60.degree. C. under reduced
pressure and further dried in vacuo to obtain 204 as a pale-tan
solid (0.824 g, 98% yield). .sup.1H NMR (500 MHz, DMSO-d.sub.6):
.delta.=7.36 (s, 2H), 6.72 (s, 2H), 3.84 (s, 6H). .sup.13C
{.sup.1H} APT NMR (126 MHz, DMSO-d6): .delta.=165.51, 156.24,
148.05, 111.99, 52.29. IR (cm.sup.-1): 3409, 3339, 3230, 1726,
1639, 1591, 1443, 1265, 996, 939, 787, 630, 543. HPLC t.sub.R=9.369
min (Method B). HRMS (m/z): 211.07213 [M+H].sup.+; Calc:
211.07133.
Preparation of Ethyl 4-amino-6-(hydroxymethyl)picolinate (205)
##STR00045##
[0187] To a refluxing suspension of 204 (0.677 g, 3.22 mmol) in
absolute EtOH (27 mL) was added NaBH.sub.4 (0.1745 g, 4.61 mmol)
portionwise over 1 h to give a pale-yellow suspension. The reaction
was then quenched with acetone (32 mL) and concentrated at
60.degree. C. under reduced pressure to a tan solid. The crude
product was dissolved in H.sub.2O (60 mL) and washed with ethyl
acetate (4.times.150 mL). The combined organics were dried over
sodium sulfate and concentrated at 40.degree. C. under reduced
pressure. Further drying in vacuo yielded 205 as a pale-yellow
solid (0.310 g, 49% yield). .sup.1H NMR (300 MHz, DMSO-d.sub.6):
.delta.=7.07 (d, J=2.1 Hz, 1H), 6.78 (m, 1H), 6.32 (s, 2H), 5.30
(t, J=5.8 Hz, 1H), 4.39 (d, J=5.6 Hz, 2H), 4.26 (q, J=7.1 Hz, 2H),
1.28 (t, J=7.1 Hz, 3H). .sup.13C APT NMR (126 MHz, DMSO-d.sub.6)
.delta.=165.57, 162.38, 155.68, 147.25, 108.50, 107.01, 63.95,
60.61, 14.24. IR (cm.sup.-1): 3439, 3217, 2974, 2917, 1717, 1643,
1600, 1465, 1396, 1378, 1239, 1135, 1022, 974, 865, 783. HPLC
t.sub.R=8.461 min (Method B). IRMS (m/z): 197.09288 [M+H].sup.+;
Calc: 197.09207.
Preparation of Ethyl 4-amino-6-(chloromethyl)picolinate (206)
##STR00046##
[0189] A mixture of thionyl chloride (2.5 mL) and 205 (0.301 g,
1.53 mmol) was stirred in an ice bath for 1 h, and then at RT for
30 min. The yellow-orange emulsion was concentrated at 40.degree.
C. under reduced pressure to an oily residue. The residue was
neutralized with sat. aq. NaHCO.sub.3 (12 mL) and then extracted
with ethyl acetate (75 mL). The organic extract was washed with
H.sub.2O (2 mL), dried over sodium sulfate, and concentrated at
40.degree. C. under reduced pressure. Further drying in vacuo gave
206 as an amber wax (0.287 g, 80% yield, corrected for residual
ethyl acetate). .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta.=7.18
(d, J=2.1 Hz, 1H), 6.78 (d, J=2.1 Hz, 1H), 6.62 (br s, 2H), 4.62
(s, 2H), 4.29 (q, J=7.1 Hz, 2H), 1.30 (t, J=7.1 Hz, 3H). .sup.13C
{.sup.1H} APT NMR (126 MHz, DMSO-d.sub.6) .delta.=164.75, 156.42,
156.19, 147.17, 109.79, 109.50, 60.97, 46.47, 14.15. IR
(cm.sup.-1): 3452, 3322, 3209, 2978, 2922, 1726, 1639, 1604, 1513,
1465, 1378, 1248, 1126, 1026, 983, 861, 783, 752, 700. HPLC
t.sub.R=12.364 min (Method B). HRMS (m/z): 215.05903 [M+H].sup.+;
Calc: 215.05818.
Preparation of Methyl
6-((1,4,10,13-tetraoxa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate
(209.2TFA.1H.sub.2O)
##STR00047##
[0191] To a clear and colorless solution of
1,7,10,16-tetraoxa-4,13-diazacyclooctadecane (7, 1.9688 g, 7.5
mmol) and diisopropylethylamine (0.8354 g, 6.5 mmol) in dry ACN
(1.075 L) at 75.degree. C. was added dropwise a solution of 206
(0.9255 g, 5.0 mmol) in dry ACN (125 mL) over 2 h 40 min. The flask
was then equipped with a condenser and drying tube, and the
slightly-yellow solution was heated at reflux for 42 h.
Subsequently, the dark-gold solution containing fine, white
precipitate was concentrated at 60.degree. C. under reduced
pressure to an amber oil. To the crude oil was added 10%
MeOH/H.sub.2O containing 0.1% TFA (10 mL). The slight suspension
was filtered, and the filtrate was purified by preparative HPLC
(Method A). Pure fractions were combined, concentrated at
60.degree. C. under reduced pressure, and then lyophilized to give
209 (1.6350 g, 50% yield) as a pale-orange solid. .sup.1H NMR (500
MHz, DMSO-d.sub.6) .delta.=8.75 (br s, 2H), 8.17-8.06 (m, 2H), 7.83
(dd, J=7.4, 1.5 Hz, 1H), 4.68 (br s, 2H), 3.91 (s, 3H), 3.85 (br t,
J=5.1 Hz, 4H), 3.69 (t, J=5.1 Hz, 4H), 3.59 (br s, 8H), 3.50 (br s,
4H), 3.23 (br t, J=5.1 Hz, 4H). .sup.13C {.sup.1H} APT NMR (126
MHz, DMSO-d.sub.6) .delta. 164.68, 158.78-157.98 (q, TFA), 151.44,
147.13, 139.01, 128.63, 124.87, 120.08-113.01 (q, TFA), 69.33,
69.00, 65.31, 64.60, 56.43, 53.29, 52.67, 46.32. .sup.19F NMR (470
MHz, DMSO-d.sub.6) .delta.=-73.84. EA Found: C, 43.88; H, 5.29; N,
6.28. Calc. for
C.sub.2H.sub.33N.sub.3O.sub.6.2CF.sub.3COOH.1H.sub.2O: C, 43.84; H,
5.67; N, 6.39. HPLC t.sub.R=12.372 min (Method B). HRMS (m/z):
412.24568 [M+H].sup.+; Calc: 412.24421.
Preparation of Ethyl
4-amino-6-((16-((6-(methoxycarbonyl)pyridin-2-yl)methyl)-1,4,10,13-tetrao-
xa-7,16-diazacyclooctadecan-7-yl)methyl)picolinate (210)
##STR00048##
[0193] Into a round-bottom flask equipped with a condenser and
drying tube were added 209 (0.4210 g, 0.64 mmol), Na.sub.2CO.sub.3
(0.3400 g, 3.2 mmol), and dry ACN (10 mL). The pale-yellow
suspension was heated to reflux over 15 min, after which 206
(0.1508 g, 0.70 mmol, corrected for residual ethyl acetate) was
added as a slight suspension in dry ACN (3.5 mL). The mixture was
heated at reflux for 44 h and then filtered. The orange filtrate
was concentrated at 60.degree. C. under reduced pressure to an
orange-brown oil (0.612 g), which was used in the next step without
further purification. HRMS (m/z): 590.32021 [M+H].sup.+; Calc:
590.31844.
Preparation of
4-Amino-6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-di-
azacyclooctadecan-7-yl)methyl)picolinic Acid (211.4TFA)
##STR00049##
[0195] Compound 210 (0.612 g) was dissolved in 6 M HCl (7 mL) and
heated at 90.degree. C. for 17 h. The orange-brown solution
containing slight precipitate was concentrated at 60.degree. C.
under reduced pressure to a pale-tan solid. To this solid was added
10% MeOH/H.sub.2O containing 0.1% TFA (3 mL). The slight suspension
was filtered and the filtrate was purified by preparative HPLC
using Method A. Pure fractions were combined, concentrated at
60.degree. C. under reduced pressure, and then lyophilized to give
211 as an off-white solid (0.2974 g, 46% yield over 2 steps).
.sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta.=8.13-8.08 (m, 2H), 7.80
(dd, J=7.3, 1.6 Hz, 1H), 7.64 (br s), 7.24 (d, J=2.3 Hz, 1H), 6.76
(d, J=2.3 Hz, 1H), 4.74 (s, 2H), 4.15 (s, 2H), 3.85 (t, J=5.0 Hz,
4H), 3.63 (t, J=5.1 Hz, 4H), 3.57-3.50 (m, 12H), 3.09 (br t, J=5.2
Hz, 4H). .sup.13C {.sup.1H} NMR (126 MHz, DMSO-d.sub.6) .delta.
165.96, 163.37, 159.47, 158.78-157.98 (q, TFA), 151.93, 151.64,
148.25, 144.68, 139.59, 128.43, 124.96, 120.79-113.68 (q, TFA),
109.40, 108.96, 70.03, 69.89, 67.09, 65.16, 57.28, 55.85, 54.47,
53.81. .sup.19F NMR (470 MHz, DMSO-d.sub.6) .delta.=-74.03. EA
Found: C, 40.60; H, 4.29; N, 7.04. Calc. for
C.sub.26H.sub.37N.sub.5O.sub.4CF.sub.3COOH: C, 40.69; H, 4.12; N,
6.98. IR (cm.sup.-1): 3387, 3161, 1735, 1670, 1204, 1130, 791, 722.
HPLC t.sub.R=11.974 min (Method B); 11.546 min (Method D). HRMS
(m/z): 548.26883 [M+H].sup.+; Calc: 548.27149.
Preparation of
6-((16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclo-
octadecan-7-yl)methyl)-4-isothiocyanatopicolinic Acid (212,
macropa-NCS)
##STR00050##
[0197] A white suspension of 211 (0.1598 g, 0.16 mmol) and
Na.sub.2CO.sub.3 (0.2540 g, 2.4 mmol) was heated at reflux in
acetone (10 mL) for 30 min before the slow addition of CSCl.sub.2
(305 .mu.L of CSCl.sub.2, 85%, Acros Organics). The resulting
orange suspension was heated at reflux for 3 h and then
concentrated at 30.degree. C. under reduced pressure to a
pale-orange solid. The solid was dissolved portionwise in 10%
ACN/H.sub.2O containing 0.2% TFA (8 mL total), filtered, and
immediately purified by preparative HPLC using Method C..sup.[246]
Pure fractions were combined, concentrated at RT under reduced
pressure to remove the organic solvent, and then lyophilized.
Fractions that were not able to be concentrated immediately were
frozen at -80.degree. C. Isothiocyanate 212 was obtained as a
mixture of white and pale-yellow solid (0.0547 g) and was stored at
-80.degree. C. in ajar of Drierite. Calculations from .sup.1H NMR
and .sup.19F NMR spectra of a sample of 212 spiked with a known
concentration of fluorobenzene estimated that 212 was isolated as a
tetra-TFA salt. .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta.=8.17-8.06 (m, 2H), 8.00 (s w/fine splitting, 1H), 7.84 (d,
J=1.5 Hz, 1H), 7.81-7.75 (d w/fine splitting, J=7.16 Hz, 1H), 4.71
(s, 2H), 4.64 (s, 2H), 3.89-3.79 (m, 8H), 3.62-3.46 (m, 16H).
.sup.19F NMR (470 MHz, DMSO-d.sub.6) .delta.=-74.17. IR
(cm.sup.-1): 3500-2800, 2083, 2026, 1735, 1670, 1591, 1448, 1183,
1130, 796, 717. HPLC t.sub.R=15.053 min (Method B); 13.885 min
(Method D). HRMS (m/z): 590.22600 [M+H].sup.+; Calc: 590.22791.
Preparation of Di-tert-butyl
(((S)-1-(tert-butoxy)-6-(3-(3-ethynylphenyl)ureido)-1-oxohexan-2-yl)carba-
moyl)-L-glutamate (214)
##STR00051##
[0199] Alkyne 214 was prepared according to published
methods.sup.[247] and isolated as an off-white powder. H NMR (500
MHz, CDCl.sub.3) .delta.=7.90 (s, 1H), 7.58 (t, 1H, J=1.7 Hz), 7.51
(dd, 1H, J.sub.1=8.2 Hz, J.sub.2=1.3 Hz), 7.18 (t, 1H, J=7.9 Hz),
7.05 (d, 1H, J=7.7 Hz), 6.38 (d, 1H, J=7.9 Hz), 6.28 (br s, 1H),
5.77 (d, 1H, J=6.9 Hz), 4.32 (m, 1H), 4.02 (m, 1H), 3.53 (m, 1H),
3.05 (m, 1H), 3.00 (s, 1H), 2.39 (m, 2H), 2.07 (m, 1H), 1.88 (m,
1H), 1.74 (m, 1H), 1.62 (m, 1H), 1.49-1.37 (m, 4H), 1.41 (s, 18H),
1.37 (s, 9H).
Preparation of 2,5-Dioxopyrrolidin-1-yl
N.sup.2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N.sup.6-(tert-butoxycarbonyl-
)-L-lysinate (215)
##STR00052##
[0201] A suspension of Fmoc-L-Lys(Boc)-OH (5.0 g, 10.7 mmol) and
N,N-disuccinimidyl carbonate (2.74 g, 10.7 mmol) in
CH.sub.2Cl.sub.2 (50 mL) was stirred at room temperature under
argon. Then DIPEA (1.86 mL, 10.7 mmol) was added, and the
suspension was stirred overnight. The solvent was evaporated under
reduced pressure and the crude product was purified by flash
chromatography (0-100% EtOAc in hexane). Lysine 215 was isolated as
a white powder (2.5 g, 41%). .sup.1H NMR (500 MHz, CDCl.sub.3)
.delta.=7.76 (d, 2H, J=7.6 Hz), 7.59 (d, 2H, J=7.3 Hz), 7.40 (t,
2H, J=7.4 Hz), 7.32 (t, 2H, J=7.3 Hz), 5.46 (br s, 1H), 4.71 (m,
2H), 4.45 (m, 2H), 4.23 (t, 1H, J=6.6 Hz), 3.14 (br s, 2H), 2.85
(s, 4H), 2.02 (m, 1H), 1.92 (m, 1H), 1.58 (m, 4H), 1.44 (s,
9H).
Preparation of tert-Butyl
N.sup.2--(N.sup.2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N.sup.6-(tert-buto-
xycarbonyl)-L-lysyl)-N.sup.6-((benzyloxy)carbonyl)-L-lysinate
(216)
##STR00053##
[0203] A suspension of L-Lys(Z)-OtBu HCl (1.49 g, 4.0 mmol) in
CH.sub.2Cl.sub.2 (15 mL) was treated with DIPEA (0.87 mL, 5.0
mmol). To the resulting mixture was added a solution of lysine 215
(2.2 g, 3.9 mmol) in CH.sub.2Cl.sub.2 (10 mL), and the reaction was
stirred overnight at room temperature under argon. It was then
washed with saturated NaCl solution, and the organic layer was
dried over MgSO.sub.4, filtered and concentrated under reduced
pressure. The crude product was purified by flash chromatography
(0-100% EtOAc in hexane), and di-lysine 216 was isolated as a white
powder (2.2 g, 72%). .sup.1H NMR (500 MHz, CDCl.sub.3) .delta.=7.76
(d, 2H, J=7.5 Hz), 7.59 (d, 2H, J=7.3 Hz), 7.40 (t, 2H, J=7.5 Hz),
7.32 (m, 8H), 6.69 (br s, 1H), 5.60 (br s, 1H), 5.06 (m, 4H), 4.72
(br s, 1H), 4.43 (m, 1H), 4.38 (m, 1H), 4.21 (m, 1H), 3.14 (m, 4H),
1.85 (m, 2H), 1.73 (m, 2H), 1.50 (m, 4H), 1.46 (s, 9H), 1.44 (s,
9H), 1.39 (m, 4H).
Preparation of 2,5-Dioxopyrrolidin-1-yl 2-(4-iodophenyl)acetate
(217)
##STR00054##
[0205] A solution of 2-(4-iodophenyl)acetic acid (786 mg, 3.0 mmol)
and EDC HCl (671 mg, 3.5 mmol) in CH.sub.2Cl.sub.2 (20 mL) was
stirred for 15 min at room temperature under argon. Then
N-hydroxysuccinimide (368 mg, 3.2 mmol) and NEt.sub.3 (0.56 mL, 4.0
mmol) were added and the reaction was stirred for 7 h. It was then
washed with saturated NaCl solution, and the organic layer was
dried over MgSO.sub.4, filtered and concentrated under reduced
pressure. The crude residue was purified by flash chromatography
(0-100% EtOAc in hexane), and the NHS ester 217 was isolated as a
white solid (760 mg, 70%). .sup.1H NMR (500 MHz, CDCl.sub.3)
.delta.=7.69 (d, 2H, J=7.9 Hz), 7.09 (d, 2H, J=7.9 Hz), 3.88 (s,
2H), 2.83 (s, 4H).
Preparation of tert-Butyl
N.sup.2--(N.sup.2-(1-azido-3,6,9,12,15,18-hexaoxahenicosan-21-oyl)-N.sup.-
6-(tert-butoxycarbonyl)-L-lysyl)-N.sup.6-((benzyloxy)carbonyl)-L-lysinate
(218)
##STR00055##
[0207] To a solution of Fmoc-protected di-lysine 216 (768 mg, 0.97
mmol) in CH.sub.2Cl.sub.2 (4 mL) was added NHEt.sub.2 (2.07 mL, 20
mmol). The solution was stirred overnight at room temperature. The
solvents were removed under reduced pressure, and the crude
product, a yellow oil, was used without further purification. To a
solution of this oil (183 mg, 0.32 mmol) in CH.sub.2Cl.sub.2 (3 mL)
were added successively solutions of NEt.sub.3 (57 .mu.L, 0.41
mmol) in CH.sub.2Cl.sub.2 (1 mL) and azido-PEG.sub.6-NHS ester (100
mg, 0.21 mmol; Broadpharm, USA) in CH.sub.2Cl.sub.2 (1 mL), and the
reaction was stirred overnight at room temperature. It was then
diluted with CH.sub.2Cl.sub.2 and washed successively with H.sub.2O
and saturated NaCl solution. The organic layer was dried over
MgSO.sub.4, filtered and concentrated under reduced pressure to
give azide 218 as a colorless oil (184 mg; 95%) without need for
further purification. Mass (ESI+): 926.4 [M+H].sup.+. Calc.
Mass=925.54.
Preparation of Di-tert-butyl
(((S)-1-(tert-butoxy)-6-(3-(3-(1-((9S,12S)-9-(tert-butoxycarbonyl)-12-(4--
((tert-butoxycarbonyl)amino)butyl)-3,11,14-trioxo-1-phenyl-2,17,20,23,26,2-
9,32-heptaoxa-4,10,13-triazatetratriacontan-34-yl)-1H-1,2,3-triazol-4-yl)p-
henyl)ureido)-1-oxohexan-2-yl)carbamoyl)-L-glutamate (219)
##STR00056##
[0209] A solution of 100 .mu.L of 0.5 M CuSO.sub.4 and 100 .mu.L of
1.5 M sodium ascorbate in DMF (0.5 mL) was mixed for 5 min and was
then added to a solution of 218 (184 mg, 0.20 mmol) and 214 (132
mg, 0.21 mmol) in DMF (2.5 mL). The resulting mixture was stirred
at room temperature for 45 min. It was then concentrated under
reduced pressure and the crude residue was purified by flash
chromatography (0-30% MeOH in EtOAc) to give triazole 219 as an
orange oil (285 mg; 87%). Mass (ESI+): 1557.2 [M+H].sup.+. Calc.
Mass=1555.90.
Preparation of Di-tert-butyl
(((S)-1-(tert-butoxy)-6-(3-(3-(1-((23S,26S)-26-(tert-butoxycarbonyl)-23-(-
4-((tert-butoxycarbonyl)amino)butyl)-33-(4-iodophenyl)-21,24,32-trioxo-3,6-
,9,12,15,18-hexaoxa-22,25,31-triazatritriacontyl)-1H-1,2,3-triazol-4-yl)ph-
enyl)ureido)-1-oxohexan-2-yl)carbamoyl)-L-glutamate (220)
##STR00057##
[0211] Cbz-Protected triazole 219 (285 mg, 0.18 mmol) was dissolved
in MeOH (15 mL) in a two-neck flask. To the solution was added 10%
Pd/C (20 mg), and the suspension was shaken and the flask
evacuated. The suspension was then placed under an H.sub.2
atmosphere and stirred overnight. It was filtered through celite,
and the filter cake was washed three times with MeOH. The combined
filtrate was concentrated under reduced pressure to give the free
amine as a colorless oil (117 mg; 45%) that was used without
further purification. Mass (ESI+): 1423.8 [M+H].sup.+. Calc.
Mass=1422.77. To a solution of the amine (117 mg, 82 .mu.mol) in
CH.sub.2Cl.sub.2 (4 mL) was added a solution of DIPEA (23 .mu.L,
131 mmol) in CH.sub.2Cl.sub.2 (1 mL), and the mixture was stirred
at room temperature under argon. Then a solution of 217 (37 mg, 103
.mu.mol) in CH.sub.2Cl.sub.2 (2 mL) was added, and the reaction was
stirred at room temperature for 2 h. It was then poured into
H.sub.2O (10 mL) and the layers were separated. The organic layer
was dried over MgSO.sub.4, filtered and concentrated under reduced
pressure to give the crude product as a colorless semi-solid. The
crude product was purified by prep TLC (10% MeOH in EtOAc) to give
phenyl iodide 220 as a colorless oil (34 mg; 25%). Mass (ESI+):
1666.6 [M+H].sup.+. Calc. Mass=1665.80.
Preparation of
(((S)-1-Carboxy-5-(3-(3-(1-((23S,26S)-26-carboxy-23-(4-(3-(2-carboxy-6-((-
16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctad-
ecan-7-yl)methyl)pyridin-4-yl)thioureido)butyl)-33-(4-iodophenyl)-21,24,32-
-trioxo-3,6,9,12,15,18-hexaoxa-22,25,31-triazatritriacontyl)-1H-1,2,3-tria-
zol-4-yl)phenyl)ureido)pentyl)carbamoyl)-L-glutamic Acid (221,
macropa-RPS-070)
##STR00058##
[0213] To a solution of 220 (34 mg, 20 .mu.mol) in CH.sub.2Cl.sub.2
(2 mL) was added TFA (0.5 mL), and the reaction was stirred at room
temperature for 5 h. It was then concentrated under reduced
pressure, and the crude product was diluted with H.sub.2O and
lyophilized to give the free amine as a TFA salt. Mass (ESI+):
1342.5 [M+H].sup.+. Mass (ESI-): 1340.6 [M-H].sup.-. Calc.
Mass=1341.50. To a solution of the amine (9 mg, 6.7 .mu.mol) in DMF
(0.5 mL) was added a solution of macropa-NCS 212 (15 mg, 25.4
.mu.mol) in DMF (0.5 mL). Then DIPEA (300 .mu.L, 1.72 mmol) was
added and the reaction was stirred at room temperature for 2 h. The
volatiles were removed under reduced pressure and the crude product
was purified by prep HPLC to give macropa-RPS-070 (221) as a white
powder (5.4 mg; 42%). Mass (ESI+): 1932.76 [M+H].sup.+. 1931.09
[M+H].sup.-. Calc. Mass=1931.91.
[0214] Preparation of Radiosynthesis of
.sup.225Ac-Macropa-RPS-070.
[0215] General.
[0216] All reagents were purchased from Sigma Aldrich unless
otherwise noted, and were reagent grade. Hydrochloric acid (HCl)
was traceSELECT.RTM. (>99.999%) for trace analysis quality.
Aluminum-backed silica thin layer chromatography (TLC) plates were
purchased from Sigma Aldrich. Stock solutions of 0.05 M HCl and 1 M
NH.sub.4OAc were prepared by dilution in Milli-Q.RTM. water.
[0217] Radiolabeling Procedure.
[0218] To a solution of .sup.225Ac(NO.sub.3).sub.3 (Oak Ridge
National Laboratory, USA) in 0.05 M HCl (17.9 MBq in 970 .mu.L) was
added 20 .mu.L of a 1 mg/mL solution of macropa-RPS-070 in DMSO.
The pH was raised to 5-5.5 by addition of 90 .mu.L M NH.sub.4OAc.
The reaction was allowed to stand at room temperature for 20 min
with periodic shaking. Then, 200 .mu.L of the reaction solution was
removed and diluted with 3.8 mL of normal saline (0.9% NaCl in
deionized H.sub.2O; VWR) to give a solution with a concentration of
910 kBq/mL. An aliquot was removed from the final solution and
spotted onto an aluminum-backed silica TLC plate to determine
radiochemical yield. An aliquot of the .sup.225Ac(NO.sub.3).sub.3
solution in 0.05M HCl was spotted in a parallel lane as a control.
The plate was immediately run in a 10% v/v MeOH/10 mM EDTA mobile
phase, and then allowed to stand for 8 h to enable radiochemical
equilibrium to be reached. The plate was visualized on a Cyclone
Plus Storage Phosphor System (Perkin Elmer) following a 3-min
exposure on the phosphor screen. The radiochemical yield was
expressed as a ratio of .sup.225Ac-macropa-RPS-070 to total
activity and was determined to be 98.1%.
[0219] Biodistribution Studies with .sup.225Ac-Macropa-RPS-070.
[0220] Cell Culture.
[0221] The PSMA-expressing human prostate cancer cell line, LNCaP,
was obtained from the American Type Culture Collection. Cell
culture supplies were from Invitrogen unless otherwise noted. LNCaP
cells were maintained in RPMI-1640 medium supplemented with 10%
fetal bovine serum (Hyclone), 4 mM L-glutamine, 1 mM sodium
pyruvate, 10 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid
(HEPES), 2.5 mg/mL D-glucose, and 50 .mu.g/mL gentamicin in a
humidified incubator at 37.degree. C./5% CO.sub.2. Cells were
removed from flasks for passage or for transfer to 12-well assay
plates by incubating them with 0.25%
trypsin/ethylenediaminetetraacetic acid (EDTA).
[0222] Inoculation of Mice with Xenografts.
[0223] All animal studies were approved by the Institutional Animal
Care and Use Committee of Weill Cornell Medicine and were
undertaken in accordance with the guidelines set forth by the USPHS
Policy on Humane Care and Use of Laboratory Animals. Animals were
housed under standard conditions in approved facilities with 12 h
light/dark cycles. Food and water was provided ad libitum
throughout the course of the studies. Hairless male nu/nu mice were
purchased from the Jackson Laboratory. For inoculation in mice,
LNCaP cells were suspended at 4.times.10.sup.7 cells/mL in a 1:1
mixture of PBS:Matrigel (BD Biosciences). Each mouse was injected
in the left flank with 0.25 mL of the cell suspension.
Biodistributions were conducted when tumors were in the range
100-400 mm.sup.3.
[0224] Biodistribution of .sup.225Ac-Macropa-RPS-070 in LNCaP
Xenograft Mice.
[0225] Fifteen LNCaP xenograft tumor-bearing mice (5 per time
point) were injected intravenously with a bolus injection of 85-95
kBq and 100 ng (50 .mu.mol) of each ligand. The mice were
sacrificed by cervical dislocation at 4, 24 and 96 h post
injection. A blood sample was removed, and a full biodistribution
study was conducted on the following organs (with contents): heart,
lungs, liver, small intestine, large intestine, stomach, spleen,
pancreas, kidneys, muscle, bone, and tumor. Tissues were weighed
and counted on a 2470 Wizard Automatic Gamma Counter (Perkin
Elmer). 1% ID/mL samples were counted prior to and following each
set of tissue samples to enable decay correction to be undertaken.
Counts were corrected for decay and for activity injected, and
tissue uptake was expressed as percent injected dose per gram (%
ID/g). Standard error measurement was calculated for each data
point.
TABLE-US-00002 TABLE 1 Organ distribution of
.sup.225Ac-macropa-RPS-070 at t = 4 h, 24 h, and 96 h following
intravenous injection in LNCaP xenograft mice (n = 5 per time
point). Values expressed as % ID/g 1 2 3 4 5 Mean SEM 4 h Blood
0.90654 0.55246 1.11808 0.8276 0.65638 0.81221 0.0986 Heart 0.75759
0.65317 0.77395 0.75148 0.6585 0.71894 0.02604 Lungs 0.99558
0.60669 1.25979 0.98587 0.88664 0.94691 0.10516 Liver 1.62187
1.34632 1.74207 1.68077 1.3957 1.55735 0.0788 Small Intestine
0.1998 0.16282 0.3104 0.24413 0.17094 0.21762 0.02721 Large
Intestine 1.36298 0.65162 1.27419 0.91656 0.81901 1.00487 0.13563
Stomach 0.33963 0.2471 0.30417 0.4109 0.21221 0.3028 0.03489 Spleen
1.40902 0.70804 1.61264 1.10815 0.8756 1.14269 0.16632 Pancreas
0.55487 0.41637 0.55317 0.4675 0.6604 0.53047 0.04182 Kidneys
65.5884 20.5274 108.233 33.654 33.0707 52.2146 15.8618 Muscle
0.68006 0.80579 0.72817 0.67666 0.65617 0.70937 0.02684 Bone
1.14861 1.12335 1.48731 0.92036 1.15463 1.16685 0.09106 Tumor
6.73177 10.7309 23.8367 15.3682 7.50352 12.8342 3.1429 24 h Blood
0.34825 0.31324 0.22083 0.29453 0.27697 0.29076 0.0211 Heart
0.52256 0.56334 0.4521 0.47914 0.46483 0.49639 0.02052 Lungs
0.53778 0.45077 0.46083 0.4286 0.44831 0.46526 0.01887 Liver
1.57844 1.47552 1.13776 1.14264 1.48473 1.38382 0.09305 Small
Intestine 0.08784 0.09914 0.08822 0.09466 0.10376 0.09473 0.00309
Large Intestine 0.13296 0.1259 0.13252 0.13425 0.13176 0.13148
0.00145 Stomach 0.1296 0.12119 0.1119 0.14675 0.15329 0.13255
0.00773 Spleen 0.62075 0.65764 0.62013 0.57685 0.58554 0.61218
0.01443 Pancreas 0.39847 0.39119 0.50347 0.33315 0.31944 0.38914
0.03252 Kidneys 4.98792 4.25707 3.94586 3.66457 4.10348 4.19178
0.22185 Muscle 0.61193 0.5149 0.44832 0.78028 0.44579 0.56025
0.06276 Bone 1.27255 1.06645 0.83943 1.00576 0.69755 0.97635
0.09828 Tumor 11.6163 9.26927 7.50158 4.41446 8.04683 8.16969
1.17583 96 h Blood 0.19042 0.19188 0.15206 0.16528 0.23822 0.18757
0.01475 Heart 0.39939 0.42398 0.42861 0.45863 0.45595 0.43331
0.01098 Lungs 0.30165 0.50912 0.46944 0.37811 0.36979 0.40562
0.03717 Liver 0.79406 0.8144 0.73301 0.7917 0.79415 0.78546 0.01374
Small Intestine 0.04372 0.0577 0.03752 0.04431 0.04136 0.04492
0.00341 Large Intestine 0.04349 0.09663 0.04522 0.04198 0.03927
0.05332 0.01087 Stomach 0.03442 0.04708 0.03448 0.02845 0.02366
0.03362 0.00393 Spleen 0.48373 0.394 0.44261 0.43481 0.53966
0.45896 0.02469 Pancreas 0.09848 0.37696 0.30549 0.31625 0.33352
0.28614 0.04847 Kidneys 1.30286 1.3239 2.00405 1.39866 1.45955
1.4978 0.12958 Muscle 0.3022 0.52492 0.25089 0.29815 0.2528 0.32579
0.05095 Bone 0.86391 0.86874 0.83831 1.12223 0.82042 0.90272
0.05557 Tumor 4.04259 4.07799 6.73954 4.58107 4.84503 4.85724
0.49449
[0226] Discussion of Results for Above-Described of
[.sup.225Ac(Macropa)].sup.+ Complexes
[0227] The in vivo stability of [.sup.225Ac(macropa)].sup.+ was
assessed by comparing its biodistribution to those of
.sup.225Ac(NO.sub.3).sub.3 and [.sup.225Ac(DOTA)].sup.-.
C.sub.57BL/6 mice were injected via tail vein with 10-50 kBq of
each radiometal complex and were sacrificed after 15 min, 1 h, or 5
h. The amount of .sup.225Ac retained in each organ was quantified
by gamma counting and reported as the percent of injected dose per
gram of tissue (% ID/g). Inadequate stability of an .sup.225Ac
complex leading to the loss of radioisotope in vivo is manifested
by the accumulation of .sup.225Ac in the liver, spleen, and bone of
mice..sup.[11,12,31] The biodistribution profile of uncomplexed
.sup.225Ac(NO.sub.3).sub.3 (FIG. 5A) reveals slow blood clearance
and excretion, coupled to large accumulation in the liver and
spleen. The biodistribution profile of [.sup.225Ac(macropa)].sup.+
(FIG. 5B) differs markedly from that of .sup.225Ac(NO.sub.3).sub.3.
[.sup.225Ac(macropa)].sup.+ was rapidly cleared from mice, with
very little activity measured in blood by 1 h post injection. Most
of the injected dose was renally excreted and subsequently detected
in the urine, which explains the moderate kidney and bladder uptake
of [.sup.225Ac(macropa)].sup.+ observed in mice at 15 min and 1 h
post injection. Of significance, [.sup.225Ac(macropa)].sup.+ did
not accumulate in any organ over the time course of the study,
indicating that the complex does not release free .sup.225Ac.sup.3+
in vivo. Its biodistribution profile was similar to that of
[.sup.225Ac(DOTA)].sup.- (FIG. 5C), which has been previously shown
to retain .sup.225Ac.sup.3+ in-vivo..sup.[7] Notably,
[.sup.225Ac(DOTA)].sup.- appeared to clear more rapidly through the
urine and was taken up to a lesser extent in the thyroid. These
differences may arise in part due to the opposite charges of the
complexes. Collectively, the results of these biodistribution
studies demonstrate that [.sup.225Ac(macropa)].sup.+ is highly
stable in vivo.
[0228] RPS-070 was conjugated to macropa-NCS, where this construct
bears a glutamate-urea-lysine moiety that inhibits the
prostate-specific membrane antigen (PSMA),.sup.[237-241] a
membrane-bound glycoprotein that is overexpressed in prostate
cancer cells.[.sup.242] An albumin-binding functional group, in
this case the group including iodophenyl, is also a critical
component of these compounds that prolongs their circulation
half-life..sup.[243,244] Radiolabeling of macropa-RPS-070 with
.sup.225Ac proceeded in 20 min at RT and pH 5-5.5 to give a RCY of
98%. .sup.225Ac-macropa-RPS-070 (85-95 kBq) was then injected into
LNCaP (prostate cancer) tumor xenograft-bearing mice, and the
biodistribution of the complex was determined at 4, 24, and 96 h
post injection (Table 1 supra, FIG. 6). .sup.225Ac-macropa-RPS-070
was rapidly cleared from the blood and primarily distributed to the
kidneys and tumor (52.+-.16% ID/g and 13.+-.3% ID/g, respectively,
at 4 h post injection). After 4 h, most of the activity cleared
from the kidneys and gradual tumor washout was observed.
Importantly, the complex exhibited negligible uptake by other
organs (<1% ID/g at 96 h post injection) and did not amass in
any organ over time. The activity that cleared from the tumor from
4-96 h remained chelated by macropa-RPS-070, as evidenced by the
lack of accumulation of .sup.225Ac in the liver, spleen, and bone
of mice during this time. These results are significant because
they demonstrate that macropa-RPS-070 can stably retain .sup.225Ac
in vivo over several days and that the construct can be selectively
targeted to tumors.
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[0284] Section 1.3
[0285] General Methods.
[0286] All solvents were purchased from Sigma Aldrich and were of
reagent grade quality unless otherwise indicated. Solvents were
dried either by distillation over an activated stainless steel
column (Pure Process Technology, LLC) column or by drying over
activated molecular sieves. Reagents were purchased from Sigma
Aldrich, except for 2-azidoacetic acid-NHS ester and the
azido-PEG.sub.n-NHS ester compounds, which were purchased from
BroadPharm. The reagents were all of reagent grade and were used
without any further purification.
[0287] All reactions described below were carried out in dried
glassware. Purifications were performed using silica chromatography
on VWR.RTM. High Purity Silica Gel 60 .ANG., preparative TLC on
silica-coated glass plates (Analtech) and by flash chromatography
using a CombiFlash Rf+(Teledyne Isco) system. Preparative HPLC was
performed using an XBridge.TM. Prep C18 5 .mu.m OBD.TM.
19.times.100 mm column (Waters) on a dual pump Agilent ProStar HPLC
fitted with an Agilent ProStar 325 Dual Wavelength UV-Vis Detector.
UV absorption was monitored at 220 nm and 280 nm. A binary solvent
system was used, with solvent A comprising H.sub.2O+0.01% TFA and
solvent B consisting of 90% v/v MeCN/H.sub.2O+0.01% TFA.
Purification was achieved using the following gradient HPLC method:
0% B 0-1 min., 0-100% B 1-28 mins., 100-0% B 28-30 mins.
[0288] Final products were identified and characterized using thin
layer chromatography, analytical HPLC and mass spectrometry. NMR
spectroscopy was used to confirm the structure of compounds 7a, 8a,
26 and 28. Analytical HPLC was performed using an XSelect.TM.
CSH.TM. C18 5 .mu.m 4.6.times.50 mm column (Waters). Mass
determinations were performed by LCMS analysis using a Waters
ACQUITY UPLC.RTM. coupled to a Waters SQ Detector 2. NMR analyses
were performed using a Bruker Avance III 500 MHz spectrometer.
Spectra are reported as ppm and are referenced to the solvent
resonances in chloroform-d (Sigma Aldrich). The purity of all
compounds evaluated in the biological assay was >95% as judged
by analytical HPLC.
di-tert-butyl(((S)-1-(tert-butoxy)-6-(3-(3-ethynylphenyl)ureido)-1-oxohexa-
n-2-yl)carbamoyl)-L-glutamate (26)
##STR00059##
[0290] Alkyne 26 was prepared according to the protocols described
in Kelly J, Amor-Coarasa A, Nikolopoulou A, Kim D, Williams C., Jr,
Ponnala S, Babich J W. Synthesis and pre-clinical evaluation of a
new class of high-affinity .sup.18F-labeled PSMA ligands for
detection of prostate cancer by PET imaging. Eur J Nucl Med Mol
Imaging 2017; 44:647-61 and isolated as an off-white powder.
.sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 7.90 (s, 1H), 7.58 (t,
1H, J=1.7 Hz), 7.51 (dd, 1H, J.sub.1=8.2 Hz, J.sub.2=1.3 Hz), 7.18
(t, 1H, J=7.9 Hz), 7.05 (d, 1H, J=7.7 Hz), 6.38 (d, 1H, J=7.9 Hz),
6.28 (br s, 1H), 5.77 (d, 1H, J=6.9 Hz), 4.32 (m, 1H), 4.02 (m,
1H), 3.53 (m, 1H), 3.05 (m, 1H), 3.00 (s, 1H), 2.39 (m, 2H), 2.07
(m, 1H), 1.88 (m, 1H), 1.74 (m, 1H), 1.62 (m, 1H), 1.49-1.37 (m,
4H), 1.41 (s, 18H), 1.37 (s, 9H).
Synthetic Procedure to that in Section 1.1 for tert-butyl
N.sup.2--(N.sup.2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N.sup.6-(tert-buto-
xycarbonyl)-L-lysyl)-N.sup.6-((benzyloxy)carbonyl)-L-lysinate
8a)
##STR00060##
[0291] 2,5-dioxopyrrolidin-1-yl
N.sup.2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N.sup.6-(tert-butoxycarbonyl-
)-L-lysinate (7a)
[0292] A suspension of Fmoc-L-Lys(Boc)-OH 6a (5.0 g, 10.7 mmol) and
N,N'-disuccinimidyl carbonate (2.74 g, 10.7 mmol) in
CH.sub.2Cl.sub.2 (50 mL) was stirred at room temperature under
argon. Then DIPEA (1.86 mL, 10.7 mmol) was added, and the
suspension was stirred overnight. The solvent was evaporated under
reduced pressure and the crude product was purified by flash
chromatography (0-100% EtOAc in hexane). The NHS ester 7a was
isolated as a white powder (2.5 g, 41%). .sup.1H NMR (500 MHz,
CDCl.sub.3) .delta. 7.76 (d, 2H, J=7.6 Hz), 7.59 (d, 2H, J=7.3 Hz),
7.40 (t, 2H, J=7.4 Hz), 7.32 (t, 2H, J=7.3 Hz), 5.46 (br s, 1H),
4.71 (m, 2H), 4.45 (m, 2H), 4.23 (t, 1H, J=6.6 Hz), 3.14 (br s,
2H), 2.85 (s, 4H), 2.02 (m, 1H), 1.92 (m, 1H), 1.58 (m, 4H), 1.44
(s, 9H).
tert-butyl
N.sup.2--(N.sup.2-(((9H-fluoren-9-yl)methoxy)carbonyl)-N.sup.6--
(tert-butoxycarbonyl)-L-lysyl)-N.sup.6-((benzyloxy)carbonyl)-L-lysinate
(8a)
[0293] A suspension of L-Lys(Z)-OtBu.HCl (1.49 g, 4.0 mmol) in
CH.sub.2Cl.sub.2 (15 mL) was treated with DIPEA (0.87 mL, 5.0
mmol). To the resulting mixture was added a solution of compound 7a
(2.2 g, 3.9 mmol) in CH.sub.2Cl.sub.2 (10 mL), and the reaction was
stirred overnight at room temperature under argon. It was then
washed with saturated NaCl solution, and the organic layer was
dried over MgSO.sub.4, filtered and concentrated under reduced
pressure. The crude product was purified by flash chromatography
(0-100% EtOAc in hexane), and di-lysine 8a was isolated as a white
powder (2.2 g, 72%). .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 7.76
(d, 2H, J=7.5 Hz), 7.59 (d, 2H, J=7.3 Hz), 7.40 (t, 2H, J=7.5 Hz),
7.32 (m, 8H), 6.69 (br s, 1H), 5.60 (br s, 1H), 5.06 (m, 4H), 4.72
(br s, 1H), 4.43 (m, 1H), 4.38 (m, 1H), 4.21 (m, 1H), 3.14 (m, 4H),
1.85 (m, 2H), 1.73 (m, 2H), 1.50 (m, 4H), 1.46 (s, 9H), 1.44 (s,
9H), 1.39 (m, 4H).
2,5-dioxopyrrolidin-1-yl 2-(4-iodophenyl)acetate (28)
##STR00061##
[0295] A solution of 2-(4-iodophenyl)acetic acid 27 (786 mg, 3.0
mmol) and EDC.HCl (671 mg, 3.5 mmol) in CH.sub.2Cl.sub.2 (20 mL)
was stirred for 15 min at room temperature under argon. Then
N-hydroxysuccinimide (368 mg, 3.2 mmol) and TEA (0.56 mL, 4.0 mmol)
were added and the reaction was stirred for 7 h. It was then washed
with saturated NaCl solution, and the organic layer was dried over
MgSO.sub.4, filtered and concentrated under reduced pressure. The
crude residue was purified by flash chromatography (0-100% EtOAc in
hexane), and the NHS ester 28 was isolated as a white solid (760
mg, 70%). .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 7.69 (d, 2H,
J=7.9 Hz), 7.09 (d, 2H, J=7.9 Hz), 3.88 (s, 2H), 2.83 (s, 4H).
[0296] Synthesis of Trifunctional Ligands (RPS-061, RPS-063,
RPS-066, RPS-067, RPS-068, RPS-069) with Representative Procedure
for Synthesis of RPS-069
##STR00062## ##STR00063##
tert-butyl
N.sup.2--(N.sup.2-(1-azido-3,6,9,12,15,18-hexaoxahenicosan-21-oyl)-N.sup.-
6-(tert-butoxycarbonyl)-L-lysyl)-N.sup.6-((benzyloxy)carbonyl)-L-lysinate
(29e)
[0297] To a solution of Fmoc-protected compound 8a (768 mg, 0.97
mmol) in CH.sub.2Cl.sub.2 (4 mL) was added diethylamine (2.07 mL,
20 mmol). The solution was stirred overnight at room temperature.
The solvents were removed under reduced pressure, and the crude
product, a yellow oil, was used without further purification. To a
solution of this yellow oil (183 mg, 0.32 mmol) in CH.sub.2Cl.sub.2
(3 mL) were added solutions of TEA (57 .mu.L, 0.41 mmol) in
CH.sub.2Cl.sub.2 (1 mL) and azido-PEG.sub.6-NHS ester (100 mg, 0.21
mmol) in CH.sub.2Cl.sub.2 (1 mL), and the reaction was stirred
overnight at room temperature. It was then diluted with
CH.sub.2Cl.sub.2 and washed successively with H.sub.2O and
saturated NaCl solution. The organic layer was dried over
MgSO.sub.4, filtered and concentrated under reduced pressure to
give azide 29e as a colorless oil (184 mg; 95%) without need for
further purification. Mass (ESI+): 926.4 [M+H].sup.+. Calc.
Mass=925.54.
di-tert-butyl
(((S)-1-(tert-butoxy)-6-(3-(3-(1-((9S,12S)-9-(tert-butoxycarbonyl)-12-(4--
((tert-butoxycarbonyl)amino)butyl)-3,11,14-trioxo-1-phenyl-2,17,20,23,26,2-
9,32-heptaoxa-4,10,13-triazatetratriacontan-34-yl)-1H-1,2,3-triazol-4-yl)p-
henyl)ureido)-1-oxohexan-2-yl)carbamoyl)-L-glutamate (30e)
[0298] A solution of 100 .mu.L 0.5M CuSO.sub.4 and 100 .mu.L 1.5 M
sodium ascorbate in DMF (0.5 mL) was mixed for 5 min and was then
added to a solution of 29e (184 mg, 0.20 mmol) and 26 (132 mg, 0.21
mmol) in DMF (2.5 mL). The resulting mixture was stirred at room
temperature for 45 min. It was then concentrated under reduced
pressure and the crude residue was purified by flash chromatography
(0-30% MeOH in EtOAc) to give triazole 30e as an orange oil (285
mg; 87%). Mass (ESI+): 1557.2 [M+H].sup.+. Calc. Mass=1555.90.
di-tert-butyl
(((S)-1-(tert-butoxy)-6-(3-(3-(1-((23S,26S)-26-(tert-butoxycarbonyl)-23-(-
4-((tert-butoxycarbonyl)amino)butyl)-33-(4-iodophenyl)-21,24,32-trioxo-3,6-
,9,12,15,18-hexaoxa-22,25,31-triazatritriacontyl)-1H-1,2,3-triazol-4-yl)ph-
enyl)ureido)-1-oxohexan-2-yl)carbamoyl)-L-glutamate (31e)
[0299] Cbz-Protected triazole 30e (285 mg, 0.18 mmol) was dissolved
in MeOH (15 mL) in a two-neck flask. To the solution was added 10%
Pd/C (20 mg), and the suspension was shaken and the flask
evacuated. The suspension was then placed under H.sub.2 atmosphere
and stirred overnight. It was filtered through celite, and the
filter cake was washed three times with MeOH. The combined filtrate
was concentrated under reduced pressure to give the free amine as a
colorless oil (117 mg; 45%) that was used without further
purification. Mass (ESI+): 1423.8 [M+H].sup.+. Calc. Mass=1422.77.
To a solution of the free amine (117 mg, 82 .mu.mol) in
CH.sub.2Cl.sub.2 (4 mL) was added a solution of DIPEA (23 .mu.L,
131 mmol) in CH.sub.2Cl.sub.2 (1 mL), and the mixture was stirred
at room temperature under argon. Then a solution of 28 (37 mg, 103
.mu.mol) in CH.sub.2Cl.sub.2 (2 mL) was added, and the reaction was
stirred at room temperature for 2 h. It was then poured into
H.sub.2O (10 mL) and the layers were separated. The organic layer
was dried over MgSO.sub.4, filtered and concentrated under reduced
pressure to give the crude product as a colorless semi-solid. The
crude product was purified by prep TLC (10% MeOH in EtOAc) to give
phenyl iodide 31e as a colorless oil (34 mg; 25%). Mass (ESI+):
1666.6 [M+H].sup.+. Calc. Mass=1665.80.
(((1S)-1-carboxy-5-(3-(3-(1-((23S,26S)-26-carboxy-33-(4-iodophenyl)-21,24,-
32-trioxo-23-(4-(3-(4-((1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraaza-
cyclododecan-2-yl)methyl)phenyl)thioureido)butyl)-3,6,9,12,15,18-hexaoxa-2-
2,25,31-triazatritriacontyl)-1H-1,2,3-triazol-4-yl)phenyl)ureido)pentyl)ca-
rbamoyl)-L-glutamic Acid (RPS-069)
[0300] To a solution of 31e (34 mg, 20 .mu.mol) in CH.sub.2Cl.sub.2
(2 mL) was added TFA (0.5 mL), and the reaction was stirred at room
temperature for 5 h. It was then concentrated under reduced
pressure and the crude product was diluted in H.sub.2O and
lyophilized to give the free amine as a TFA salt. Mass (ESI+):
1342.5 [M+H].sup.+. Mass (ESI-): 1340.6 [M-H].sup.-. Calc.
Mass=1341.50. To a solution of p-SCN-Bn-DOTA.2.5HCl.2.5H.sub.2O
(Macrocyclics, Inc.) (13 mg, 19 .mu.mol) in H.sub.2O (0.5 mL) was
added a solution of the free amine (18 mg, 13 .mu.mol) in DMF (1
mL). DIPEA was added until the reaction was pH 9). The reaction was
stirred at room temperature for 3 h, at which point the reaction
mixture was then purified by prep HPLC. The peak corresponding to
the desired product was collected and lyophilized to give RPS-069
as a white powder (8 mg; 32%). Mass (ESI+): 1893.3 [M+H].sup.+,
947.6 [(M+2H)/2].sup.+. Mass (ESI-): 1891.4 [M-H].sup.-, 945.5
[(M-2H)/2]. Calc. Mass=1892.70.
(((1S)-1-carboxy-5-(3-(3-(1-((17S,20S)-20-carboxy-27-(4-iodophenyl)-15,18,-
26-trioxo-17-(4-(3-(4-((1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraaza-
cyclododecan-2-yl)methyl)phenyl)thioureido)butyl)-3,6,9,12-tetraoxa-16,19,-
25-triazaheptacosyl)-1H-1,2,3-triazol-4-yl)phenyl)ureido)pentyl)carbamoyl)-
-L-glutamic Acid (RPS-061)
[0301] RPS-061 was synthesized from the common building blocks 26,
8a and 28 and azido-PEG.sub.4-NHS ester according to the procedure
described for RPS-069. Mass (ESI+): 1805.6664 [M+H].sup.+. Calc.
Mass=1804.6594.
(((1S)-1-carboxy-5-(3-(3-(1-((14S,17S)-17-carboxy-24-(4-iodophenyl)-12,15,-
23-trioxo-14-(4-(3-(4-((1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraaza-
cyclododecan-2-yl)methyl)phenyl)thioureido)butyl)-3,6,9-trioxa-13,16,22-tr-
iazatetracosyl)-1H-1,2,3-triazol-4-yl)phenyl)ureido)pentyl)carbamoyl)-L-gl-
utamic Acid (RPS-063)
[0302] RPS-063 was synthesized from the common building blocks 26,
8a and 28 and azido-PEG.sub.3-NHS ester according to the procedure
described for RPS-069. Mass (ESI+): 1762.4 [M+H].sup.+. Mass
(ESI-): 1760.5 [M-H].sup.-. Calc. Mass=1761.71.
(((1S)-1-carboxy-5-(3-(3-(1-((29S,32S)-32-carboxy-39-(4-iodophenyl)-27,30,-
38-trioxo-29-(4-(3-(4-((1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraaza-
cyclododecan-2-yl)methyl)phenyl)thioureido)butyl)-3,6,9,12,15,18,21,24-oct-
aoxa-28,31,37-triazanonatriacontyl)-1H-1,2,3-triazol-4-yl)phenyl)ureido)pe-
ntyl)carbamoyl)-L-glutamic Acid
[0303] (RPS-066) RPS-066 was synthesized from the common building
blocks 26, 8a and 28 and azido-PEGs-NHS ester according to the
procedure described for RPS-069. Mass (ESI+): 1982.3 [M+H].sup.+,
991.5 [(M+2H)/2].sup.-. Calc. Mass=1980.76.
(((1S)-1-carboxy-5-(3-(3-(1-((41S,44S)-44-carboxy-51-(4-iodophenyl)-39,42,-
50-trioxo-41-(4-(3-(4-((1,4,7,10-tetrakis(carboxymethyl)-1,4,7,10-tetraaza-
cyclododecan-2-yl)methyl)phenyl)thioureido)butyl)-3,6,9,12,15,18,21,24,27,-
30,33,36-dodecaoxa-40,43,49-triazahenpentacontyl)-1H-1,2,3-triazol-5-yl)ph-
enyl)ureido)pentyl)carbamoyl)-L-glutamic Acid (RPS-067)
[0304] RPS-067 was synthesized from the common building blocks 26,
8a and 28 and azido-PEG.sub.12-NHS ester according to the procedure
described for RPS-069. Mass (ESI+): 1079.7 [(M+2H)/2].sup.+. Mass
(ESI-): 2155.6 (M-H).sup.-, 1077.7 [(M-2H)/2].sup.-. Calc.
Mass=2156.86.
[0305] Synthesis of RPS-068
##STR00064## ##STR00065##
tert-butyl
N.sup.2--(N.sup.2-(2-azidoacetyl)-N.sup.6-(tert-butoxycarbonyl)-L-lysyl)--
N.sup.6-((benzyloxy)carbonyl)-L-lysinate (32)
[0306] To a solution of Fmoc-protected 8a (768 mg, 0.97 mmol) in
CH.sub.2Cl.sub.2 (4 mL) was added diethylamine (2.07 mL, 20 mmol).
The solution was stirred overnight at room temperature. The
solvents were removed under reduced pressure, and the crude
product, the free amine as a yellow oil, was used without further
purification. To a solution of free amine (356 mg, 0.63 mmol) in
CH.sub.2Cl.sub.2 (6 mL) was added a solution of TEA (175 .mu.L,
1.26 mmol) in CH.sub.2Cl.sub.2 (1 mL), and the resulting mixture
was stirred at room temperature. Then a solution of 2-azidoacetic
acid NHS ester (138 mg, 0.69 mmol) in CH.sub.2Cl.sub.2 (3 mL) was
added, and the reaction was stirred at room temperature. After 3 h,
it was diluted with CH.sub.2Cl.sub.2 and washed successively with
H.sub.2O and saturated NaCl solution. The organic layer was dried
over MgSO.sub.4, filtered and concentrated under reduced pressure
to give pale yellow azide 32 (374 mg, 920%) that was used without
further purification. Mass (ESI+): 648.1 [M+H].sup.+. Calc.
Mass=647.36.
di-tert-butyl
(((S)-1-(tert-butoxy)-6-(3-(3-(1-((9S,12S)-9-(tert-butoxycarbonyl)-12-(4--
((tert-butoxycarbonyl)amino)butyl)-3,11,14-trioxo-1-phenyl-2-oxa-4,10,13-t-
riazapentadecan-15-yl)-11H-1,2,3-triazol-5-yl)phenyl)ureido)-1-oxohexan-2--
yl)carbamoyl)-L-glutamate (33)
[0307] A solution of 150 .mu.L 0.5M CuSO.sub.4 and 150 .mu.L 1.5M
sodium ascorbate in DMF (0.2 mL) was mixed for 5 min and was then
added to a solution of azide 32 (374 mg, 0.54 mmol) and alkyne 26
(358 mg, 0.54 mmol) in DMF (2 mL). The mixture was stirred at room
temperature for 2 h before the solvent was removed under reduced
pressure. The resulting residue was dissolved in CH.sub.2Cl.sub.2
and washed with H.sub.2O. The organic layer was dried over
MgSO.sub.4, filtered and concentrated under reduced pressure. The
crude product was purified by flash chromatography (0-10% MeOH in
EtOAc), but a small impurity remained. Therefore a second
purification was performed by prep TLC (100% EtOAc), and the
product was isolated as a colorless oil (146 mg; 21%). Mass (ESI+):
1278.6 [M+H].sup.+. Calc. Mass=1277.73.
di-tert-butyl
(((S)-1-(tert-butoxy)-6-(3-(3-(1-(2-(((10S,13S)-13-(tert-butoxycarbonyl)--
20-(4-iodophenyl)-2,2-dimethyl-4,11,19-trioxo-3-oxa-5,12,18-triazaicosan-1-
0-yl)amino)-2-oxoethyl)-1H-1,2,3-triazol-5-yl)phenyl)ureido)-1-oxohexan-2--
yl)carbamoyl)-L-glutamate (34)
[0308] Triazole 33 (146 mg, 0.11 mmol) was dissolved in MeOH (10
mL) in a two-neck flask. To the solution was added 10% Pd/C (10
mg), and the suspension was shaken while the flask was evacuated.
Then the suspension was stirred under H.sub.2 atmosphere for 2 h
before the mixture was filtered through celite. The filter cake was
washed three times with MeOH and the filtrates were combined and
concentrated under reduced pressure to give the free amine as a
black residue (91 mg; 72%) that contained traces of minor
impurities. The crude product was used without further
purification. Mass (ESI+): 1144.6 [M+H].sup.+. Calc. Mass=1143.69.
To a solution of free amine (90 mg, 79 .mu.mol) and NEt.sub.3 (14
.mu.L, 150 .mu.mol) in CH.sub.2Cl.sub.2 (4 mL) was added a solution
of 28 (36 mg, 100 .mu.mol) in CH.sub.2Cl.sub.2 (1 mL). The
resulting mixture was stirred overnight at room temperature, then
it was diluted with CH.sub.2Cl.sub.2 and washed successively with
H.sub.2O and saturated NaCl solution. The organic layer was dried
over MgSO.sub.4, filtered and concentrated under reduced pressure
to give a black residue. The residue was dissolved in EtOAc, and a
black precipitate was removed by filtration. The resulting crude
product was purified by prep TLC (5% MeOH in EtOAc), and phenyl
iodide 34 was isolated as a white solid (21 mg; 19%). Mass (ESI+):
1388.4 [M+H].sup.+. Calc. Mass=1387.63.
(((1S)-1-carboxy-5-(3-(3-(1-(2-(((2S)-1-(((S)-1-carboxy-5-(2-(4-iodophenyl-
)acetamido)pentyl)amino)-1-oxo-6-(3-(4-((1,4,7,10-tetrakis(carboxymethyl)--
1,4,7,10-tetraazacyclododecan-2-yl)methyl)phenyl)thioureido)hexan-2-yl)ami-
no)-2-oxoethyl)-1H-1,2,3-triazol-5-yl)phenyl)ureido)pentyl)carbamoyl)-L-gl-
utamic Acid (RPS-068)
[0309] To a solution of 34 (20 mg, 15 mol) in CH.sub.2Cl.sub.2 (3.5
mL) was added TFA (0.5 mL). The reaction was stirred at room
temperature for 4 h, then it was concentrated under reduced
pressure. The crude residue was dissolved in H.sub.2O and
lyophilized to give the free amine as a TFA salt. Mass (ESI+):
1064.1 [M+H].sup.+. Calc. Mass=1063.33. To a solution of
p-SCN-Bn-DOTA.2.5HCl.2.5H.sub.2O (Macrocyclics, Inc.) (10 mg, 15
.mu.mol) in 1 mL 50% DMF in H.sub.2O was added a solution of free
amine (16 mg, 15 .mu.mol) in DMF (0.7 mL). NEt.sub.3 was added (110
.mu.L) until the pH of the reaction was approximately 9. The
reaction was stirred for 1 h, then the reaction mixture was
purified by prep HPLC. The peak corresponding to the product was
collected and lyophilized to give RPS-068 as a white powder (2.4
mg; 10%). Mass (ESI+): 1615.2 [M+H].sup.+. Mass (ESI-): 1613.3
[M-H].sup.-, 806.4 [(M-2H)/2].sup.-. Calc. Mass=1614.53.
[0310] Radiochemistry
[0311] General Methods:
[0312] All reagents were purchased from Sigma Aldrich unless
otherwise noted, and were reagent grade. Hydrochloric acid (HCl)
and sodium acetate (NaOAc) were of traceSELECT.RTM. (>99.999%)
quality. All water (H.sub.2O) used was highly pure (18 m.OMEGA.).
Analytical IPLC was performed on a dual-pump Varian Dynamax IPLC
(Agilent Technologies) fitted with a dual UV-Vis detector, and
radiochemical purity was determined using a NaI(Tl) flow count
detector (Bioscan). UV absorption was monitored at 220 nm and 280
nm. Solvent A was 0.01% trifluoroacetic acid (TFA) in H.sub.2O and
solvent B was 0.01% TFA in 90% v/v acetonitrile (MeCN):H.sub.2O.
Analyses were performed on a Symmetry C18 4.6.times.50 mm, 100-A
column (Waters) at a flow rate of 2 mL/min and a gradient of 0% B
to 100% B over 10 minutes.
[0313] Production of Ga-66:
[0314] Gallium-66 (t.sub.1/2=9.4 h) was produced from the
irradiation of a natural zinc target (Alfa Aesar; 0.5 g, 100 .mu.m
thickness, 99.999%) by a (p,n) reaction over 2 h using a 15 MeV
beam and a 17.5 mA current. The irradiation of natural zinc
produces Ga-66, Ga-67 and Ga-68. The target was left overnight to
allow Ga-68 (t.sub.1/2=68 min) to decay before processing. The
principal radionuclidic impurity during processing was Ga-67
(t.sub.1/2=78.3 h), at approximately 3%. The target was dissolved
in conc. HCl (5 mL) and the .sup.66Ga.sup.3+ ions were separated
from Zn.sup.2+ ions by 20 mg UTEVA anion exchange (Eichrom)
according to previously published methods [20]. The column was
later washed twice with 3 ml of a 5M HCl solution to eliminate the
excess Zn.sup.2+. Finally, the purified .sup.66Ga.sup.3+ ions were
eluted with H.sub.2O (0.5 mL), leading to a final solution
containing 2.14-2.36 GBq/mL (58-64 mCi/mL) and approximately 0.1 M
HCl.
[0315] Radiolabeling of RPS Series:
[0316] .sup.66Ga-Labeled ligands were prepared according to the
following procedure. 100 .mu.L of the Ga-66 stock solution
containing 167-205 MBq (4.5-5.5 mCi) was diluted with 1 mL 0.05M
HCl. To this solution was added 40-80 .mu.L of a 1 mg/mL solution
of precursor in DMSO. The reaction was initiated by addition of 40
.mu.L 3N NaOAc, and the solution was mixed at 95.degree. C. on an
Eppendorf ThermoMixer.RTM. C (VWR) for 25 min. The mixture was then
diluted with H.sub.2O and passed through a pre-activated Sep-Pak
C18 Plus Light cartridge (Waters). The cartridge was washed with
H.sub.2O and the product was eluted with 100 .mu.L EtOH (300 proof,
VWR) followed by 900 .mu.L saline (0.9% NaCl solution; VWR). Final
radioactivity concentrations were in the range 7.4-85 MBq/mL
(0.2-2.3 mCi/mL), and radiochemical purity was greater than
90%.
[0317] Labeling with Lu-177:
[0318] No-carrier-added Lu-177 (EndolucinBeta.RTM.) was purchased
from iTG (Garching, Germany) as the chloride salt, with an activity
at calibration of 1.5-3.0 GBq (40-80 mCi). An aliquot containing
0.52-0.93 GBq (14-25 mCi) of the Lu-177 stock solution was diluted
to 1 mL with 0.05M HCl. To this solution was added 20 .mu.g of
precursor as a 1 mg/mL solution in DMSO. The reaction was initiated
by raising the pH to 4-5 using 3N NaOAc (20-30 .mu.L). The buffered
solution was heated for 10 min at 95.degree. C. on an analog
heating block (VWR). After the solution had cooled to room
temperature, it was diluted with H.sub.2O (9 mL) and passed through
a pre-activated Sep-Pak C18 Plus Light cartridge (Waters). The
cartridge was washed with H.sub.2O (5 mL) and the product was
eluted with 500 .mu.L EtOH (200 proof, VWR) followed by 500 .mu.L
saline (0.9% NaCl solution; VWR). An aliquot (40-98 .mu.L) was
removed from this solution and diluted to 4 mL with saline. The
final concentration of each ligand in the injected solution was
0.23-0.28 .mu.M, with a range of activity of 3.5-8.8 MBq/mL (93-240
.mu.Ci/mL). The specific activity of the .sup.177Lu-labeled
compounds ranged from 15.8-48.8 GBq/.mu.mol. Radiochemical yields
were 33-80% after purification and reformulation, and radiochemical
purity was greater than 98%.
[0319] Cell Culture:
[0320] The PSMA expressing human prostate cancer cell line, LNCaP,
was obtained from the American Type Culture Collection. Cell
culture supplies were obtained from Invitrogen unless otherwise
noted. LNCaP cells were maintained in RPMI-1640 medium supplemented
with 10% fetal bovine serum (Hyclone), 4 mM L-glutamine, 1 mM
sodium pyruvate, 10 mM
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 2.5
mg/mL D-glucose, and 50 .mu.g/mL gentamicin in a humidified
incubator at 37.degree. C./5% CO.sub.2. Cells were removed from
flasks for passage or for transfer to 12-well assay plates by
incubating them with 0.25% trypsin/ethylenediaminetetraacetic acid
(EDTA).
[0321] In Vitro Determination of IC.sub.50:
[0322] IC.sub.50 values of the non-labeled, metal-free ligands were
determined by screening in a multi-concentration competitive
binding assay against
.sup.99mTc-((7S,12S,16S)-1-(1-(carboxymethyl)-1H-imidazol-2-yl)-2-((1-(ca-
rboxymethyl)-1H-imidazol-2-yl)methyl)-9,14-dioxo-2,8,13,15-tetraazaoctadec-
ane-7,12,16,18-tetracarboxylic acid technetium tricarbonyl complex)
(.sup.99mTc-MIP-1427) for binding to PSMA on LNCaP cells, according
to previously described methods [18,19] with small modifications.
Briefly, LNCaP cells were plated 48 h prior to the experiment to
achieve a density of approximately 5.times.10.sup.5 cells/well (in
triplicate) in RPMI-1640 medium supplemented with 0.25% bovine
serum albumin. The cells were incubated for 2 h with 1 nM
.sup.99mTc-MIP-1427 in serum-free RPMI-1640 medium in the presence
of 0.001-10,000 nM test compounds. Radioactive incubation media was
then removed by pipette and the cells were washed twice using 1 mL
ice-cold PBS 1.times. solution. Cells were harvested from the
plates following treatment with 1 mL 1M NaOH and transferred to
tubes for radioactive counting using a 2470 Wizard.sup.2 Automatic
Gamma Counter (Perkin Elmer). Standard solutions (10% of activity
added to each well) were prepared to enable decay correction.
IC.sub.50 values were determined by fitting the data points to a
sigmoidal Hills1 curve in Origin software.
[0323] Inoculation of Mice with Xenografts:
[0324] All animal studies were approved by the Institutional Animal
Care and Use Committee of Weill Cornell Medicine and were
undertaken in accordance with the guidelines set forth by the USPHS
Policy on Humane Care and Use of Laboratory Animals. Animals were
housed under standard conditions in approved facilities with 12 h
light/dark cycles. Food and water was provided ad libitum
throughout the course of the studies. Hairless male nu/nu mice were
purchased from the Jackson Laboratory. For inoculation in mice,
LNCaP cells were suspended at 4.times.10.sup.7 cells/mL in a 1:1
mixture of PBS:Matrigel (BD Biosciences). Each mouse was injected
in the left flank with 0.25 mL of the cell suspension. The mice
were imaged when the tumors reached approximately 200-400 mm.sup.3,
while biodistributions were conducted when tumors were in the range
100-400 mm.sup.3.
[0325] Imaging of .sup.66Ga-RPS Ligands in LNCaP Xenograft
Mice:
[0326] LNCaP xenograft tumor-bearing mice (2-3 per compound)
injected intravenously with a bolus injection of 0.56-5.4 MBq
(15-145 .mu.Ci) of the .sup.66Ga-labeled ligand. The specific
activity of the tracers was in the range 14.8-47 MBq/.mu.mol
(0.4-1.27 mCi/.mu.mol). The mice were imaged using PET/CT
(Inveon.TM.; Siemens Medical Solutions, Inc.) at 1, 3, 6 and 24 h
post-injection following inhalation anesthetization with
isoflurane. Total acquisition time was 30 min for the 1 h, 3 h and
6 h images, and 60 min for 24 h time point. A CT scan was obtained
immediately before the acquisition for both anatomical
co-registration and attenuation correction. Images were
reconstructed using the Inveon.TM. software supplied by the vendor.
Image-derived tumor and kidney uptake was estimated by comparison
to a 10% injected dose per cubic mm (% ID/mm.sup.3) standard
introduced into the imaging field of view. The standard was
prepared by dilution of 10% of the injected activity to 1 mL with
saline. Volumes of interest (VOIs) were drawn with the aid of the
CT and confirmed by PET. The contents of the VOIs were integrated
and the calculated counts were converted to % ID/mm.sup.3 by direct
comparison to the aforementioned standard following correction for
activity injected.
[0327] Biodistribution Studies of 177Lu-Labeled Ligands in LNCaP
Xenograft Mice:
[0328] LNCaP xenograft tumor-bearing mice (5 per time point per
compound) were injected intravenously with a bolus injection of
348-851 kBq (9.4-23 .mu.Ci) and 37-50 ng (23-25 .mu.mol) of each
ligand. The mice were sacrificed at 4, 24 and 96 h post injection.
A blood sample was removed, and a full biodistribution study was
conducted on the following organs (with contents): heart, lungs,
liver, small intestine, large intestine, stomach, spleen, pancreas,
kidneys, muscle, bone and tumor. Tissues were weighed and counted
on a 2470 Wizard.sup.2 Automatic Gamma Counter (Perkin Elmer).
Counts were corrected for decay and for activity injected, and
tissue uptake was expressed as percent injected dose per gram (%
ID/g). Standard error measurement was calculated for each data
point.
[0329] Dosimetry:
[0330] The dosimetry was calculated assuming a linear interpolation
between the three time points. The average injected dose per organ
was calculated by using the average of the activity and organ
weights of the mice at that time point. Intermediate time points at
every 4 hours were generated using the linear approximation and all
time points were corrected for decay during the time interval
between points. These curves were integrated using a trapezoidal
approximation and the sum used to determine the residence time.
[0331] Statistical Analysis:
[0332] A comprehensive statistical analysis was performed to
compare the tissue uptake of each compound across time. The
normality assumption was visually checked by a quantile-quantile
(QQ) plot, and a log transformation was applied to the data to
remove the skew effect. Under each organ and each compound, a
one-way ANOVA (Analysis of Variance) with Tukey's honestly
significant difference (HSD) post-hoc test was used to evaluate the
difference in measurement across three time points. An overall
P-value under an F-test and pairwise ones under t-test was
determined. Furthermore, a two-way ANOVA was used to assess the
influence of time, compound and their interaction in each organ.
The P-values are reported. A confidence interval of 95% was used to
determine statistical significance.
[0333] Section 1.3 Results and Discussion
[0334] The three moieties were linked by an azide-derivatized
polyethyleneglycol (PEG) spacer incorporating 0 (RPS-068), 3
(RPS-063), 4 (RPS-061), 6 (RPS-069), 8 (RPS-066) or 12 (RPS-067)
PEG subunits (see Table 2). No degradation or decomposition of the
ligands was observed over the course of three months during storage
at 4.degree. C. as determined by analytical HPLC. In contrast,
similar analogues in which a Gly-Gly-Gly linker or a
C.sub.7H.sub.14 linker was used in place of the PEG spacer were
found to decompose over the course of a few weeks under the same
storage conditions.
[0335] In an effort to minimize the use of animals, an initial
screening of the compounds was performed using .mu.PET/CT imaging
to avoid unnecessary testing. For this purpose, Ga-66 was selected
in preference to other PET radionuclides such as Ga-68 or Sc-44 due
to its longer half-life (t.sub.1/2=9.4 h) and the possibility of
producing larger quantities (>1.85 GBq/50 mCi) in the cyclotron.
Greater than 99% of the Ga-66 was recovered in the purification
process, but labeling yields remained consistently low
(46.4.+-.20.5%, n=7). The variable and low labeling yields were
likely due to the presence of Zn.sup.2+ ions in the labeling
reaction due to incomplete separation of the .sup.66Ga.sup.3+ ions
from the dissolved target material.
[0336] The .sup.177Lu-labeled constructs were prepared in 67.+-.17%
(n=20) radiochemical yield following purification and
reformulation. Variation in final product yield was largely due to
differences in trapping efficiency by the C18 cartridge, with
.sup.177Lu-RPS-067 and .sup.177Lu-RPS-068 showing the lowest
trapping (approximately 40%). Labeling yields prior to purification
were typically >75% for all ligands as determined by radioHPLC.
There was no apparent correlation between PEG length and labeling
yield. The Lu-177 labeled ligands were stable to radiolysis for 24
h when stored at 4.degree. C. Radiochemical stability was not
determined at room temperature.
[0337] The total amount of ligand injected was 22-24 .mu.mol per
mouse in order to remain proportional to clinical mass doses of
.sup.177Lu-PSMA-617 administered to human subjects [4]. The
specific activity of the preparations ranged from 15.8-48.8
GBq/.mu.mol, consistent with the values reported for the
preclinical evaluation of .sup.177Lu-PSMA-617 [21]. The mass of
.sup.66Ga-labeled ligands injected was 4 .mu.g per mouse,
corresponding to 1.8-2.5 nmol. This greater mass was required to
account for the poorer labeling yields with this radionuclide.
[0338] All compounds were evaluated for PSMA binding in vitro using
a cell-based competitive binding assay. All compounds were highly
potent (IC.sub.50<10 nM), validating our selection of the
3-ethynylphenylurea derivative of Glu-urea-Lys as the
PSMA-targeting pharmacophore. The range of affinities was defined
by RPS-063 (IC.sub.50=1.5 0.3 nM) and RPS-067 (IC.sub.50=9.5 1.1
nM) (Table 2). Potency generally decreased with increasing PEG
linker length, although RPS-068 (PEG.sub.0; IC.sub.50=2.1.+-.0.1
nM) was slightly less potent than RPS-063. In the same assay the
IC.sub.50 of PSMA-617 was determined to be 6.6.+-.0.7 nM (Table 2),
consistent with the previously reported value [21].
TABLE-US-00003 TABLE 2 Summary of compound structures and key in
vitro and in vivo characteristics. IC.sub.50 values were determined
by a competitive binding assay in LNCaP cells. Tumor uptake was
determined by biodistribution studies with the corresponding
.sup.177Lu-labeled compound in LNCaP xenograft tumor-bearing mice.
(a = 4 h p.i.; b = 24 h p.i.) Max. Mol. Tumor Wt. IC.sub.50
Uptake.sup.a Cpd. PEG Structure (g/mol) (nM) (% ID/g) PSMA- 617
n.a. ##STR00066## 1042.15 6.6 .+-. 0.7 14.4 .+-. 2.5 RPS- 068 0
##STR00067## 1615.52 2.1 .+-. 0.1 26.9 .+-. 2.0.sup.b ##STR00068##
Max. Mol. Tumor Wt. IC.sub.50 Uptake.sup.a Cpd. PEG Structure
(g/mol) (nM) (% ID/g) RPS- 063 3 n = 2 1761.71 1.5 .+-. 0.3 30.0
.+-. 6.9 RPS- 061 4 n = 3 1805.76 4.1 .+-. 0.9 20.4 .+-. 3.1 RPS-
069 6 n = 5 1893.87 3.8 .+-. 0.4 17.0 .+-. 2.1 RPS- 066 8 n = 7
1981.97 5.2 .+-. 0.4 18.7 .+-. 1.1 RPS- 067 12 n = 11 2158.18 9.5
.+-. 1.1 7.6 .+-. 1.2
[0339] Preliminary screening of the compounds in mice was performed
by .mu.PET/CT imaging using .sup.66Ga (FIG. 7) with the intention
of avoiding full biodistribution studies on compounds that showed
poor targeting. Images were analyzed to determine quantitative
uptake in the tumor and kidneys at 1, 3, 6 and 24 h post injection.
In the tumor, uptake was high but decreased with increasing PEG
length. .sup.66Ga-RPS-068 (PEG0; maximum uptake of 9.7.+-.2.0%
ID/cm.sup.3 (3 h); 8.3.+-.2.8% ID/cm.sup.3 at 24 h) and
.sup.66Ga-RPS-063 (PEG3; maximum uptake of 9.5.+-.2.4% ID/cm.sup.3
(6 h); 7.9.+-.3.0% ID/cm.sup.3 at 24 h) showed the greatest uptake,
with .sup.66Ga-RPS-061 (PEG4; 6.1.+-.1.1% ID/cm.sup.3) and
.sup.66Ga-RPS-069 (PEG6; 7.0.+-.3.9% ID/cm.sup.3) demonstrating
comparable uptake at 24 h post injection. .sup.66Ga-RPS-066 (PEG8;
maximum uptake of 7.8.+-.0.7% ID/cm.sup.3 (3 h); 5.5.+-.0.4%
ID/cm.sup.3 at 24 h) and .sup.66Ga-RPS-067 (PEG12; maximum uptake
of 6.6.+-.3.2% ID/cm.sup.3 (1 h); 3.1.+-.1.7% ID/cm.sup.3 at 24 h)
showed lower uptake at all time points, but still exceeded
.sup.66Ga-PSMA-617 (maximum uptake of 3.1.+-.0.4% ID/cm.sup.3 (1
h); 1.1.+-.0.4% ID/cm.sup.3 at 24 h. Kidney uptake was generally on
the same order as tumor uptake and was greatest at 1 h post
injection. Uptake ranged from 10.5.+-.2.1% ID/cm.sup.3
(.sup.66Ga-RPS-061) to 3.7.+-.0.5% ID/cm.sup.3 (.sup.66Ga-RPS-067)
at 1 h post injection, and from 1.9.+-.0.3% ID/cm.sup.3
(.sup.66Ga-RPS-068) to 0.2.+-.0.1% ID/cm.sup.3 (.sup.66Ga-RPS-066
and .sup.66Ga-RPS-067) at 24 h post injection. In comparison, the
maximum kidney uptake of .sup.66Ga-PSMA-617 was 0.4.+-.0.1%
ID/cm.sup.3, while uptake at 24 h was 0.1.+-.0.1% ID/cm.sup.3.
[0340] Following the promising imaging studies, biodistribution
studies of the .sup.177Lu-labeled ligands confirmed the trends
evident in the PET images. Although the affinity of the compounds
for PSMA is clustered within one order of magnitude, the tissue
distribution of the ligands showed considerable variation. This was
most evident in the tissues that are known to express PSMA,
including the tumor and the kidney (FIG. 8). Tumor uptake was high
and remained high for .sup.177Lu-RPS-068 (PEG.sub.0),
.sup.177Lu-RPS-063 (PEG.sub.3), .sup.177Lu-RPS-061 (PEG.sub.4),
.sup.177Lu-RPS-069 (PEG.sub.6) and .sup.177Lu-RPS-066 (PEG). For
the higher affinity compounds .sup.177Lu-RPS-068 and
.sup.177Lu-RPS-063, uptake at 4 h p.i. was 21.8.+-.2.8% ID/g and
30.0.+-.3.1% ID/g, respectively, with 14.9.+-.1.5% ID/g and
12.9.+-.0.5% ID/g still remaining at 96 h p.i. Clearance was not
statistically significant by 24 h (p>0.13). Uptake of
.sup.177Lu-RPS-069 and .sup.177Lu-RPS-066 was 17.0.+-.2.1% ID/g and
18.7.+-.1.1% ID/g respectively at 4 h p.i. and decreased to
9.8.+-.0.8% ID/g and 5.9.+-.0.7% ID/g at 96 h p.i. Nevertheless,
these uptake values are significantly greater after 24 h p.i. than
those observed for .sup.177Lu-PSMA-617 (14.4.+-.1.1% ID/g and
3.5.+-.0.3% ID/g at 4 h and 96 h p.i., p<0.001).
.sup.177Lu-RPS-067 (PEG.sub.12), the lowest affinity ligand,
accumulated at only 7.6.+-.1.2% ID/g at 4 h p.i. and had cleared to
3.2.+-.0.1% ID/g at 96 h p.i. This uptake was significantly lower
than all other ligands (p<0.001) except .sup.177Lu-PSMA-617.
[0341] A similar trend within the RPS series was observed for
kidney uptake, with the lowest affinity ligand, .sup.177Lu-RPS-067,
distinguished by significantly lower uptake (54.9.+-.13.2% ID/g) at
4 h p.i. than the other RPS ligands tested (p<0.004) (FIG. 8).
Kidney uptake exceeded 100% ID/g at 4 h p.i. for all other ligands
of the RPS series, while .sup.177Lu-PSMA-617 was found to clear
rapidly (14.1.+-.3.1% ID/g at 4 h p.i.) in agreement with published
reports [21]. Prolonged retention of .sup.177Lu-RPS-068
(87.3.+-.6.7% ID/g at 24 h p.i.) and .sup.177Lu-RPS-063
(51.8.+-.8.6% ID/g at 24 h p.i.) was evident, but
.sup.177Lu-RPS-066 (6.2.+-.0.8% ID/g at 24 h p.i.) and
.sup.177Lu-RPS-067 (4.6.+-.0.6% ID/g at 24 h p.i.) cleared
significantly (p<0.001) and more rapidly. Uptake of these two
ligands was significantly lower than the other RPS ligands
(p<0.001) but not significantly different to each other
(p<0.14).
[0342] In combination with persistent tumor accumulation, more
rapid kidney clearance gave rise to tumor-to-kidney ratios of
1.92.+-.0.30 and 1.25.+-.0.20 for .sup.177Lu-RPS-066 and
.sup.177Lu-RPS-067 at 24 h p.i. These ratios are significantly
higher than the other RPS ligands (p<0.001), but reflect low and
rapid kidney clearance rather than high and persistent tumor
uptake. For the same reason, the tumor-to-kidney ratio of
.sup.177Lu-PSMA-617 is significantly higher than the other ligands
at all time points studied (p<0.001). By 96 h, each member of
the RPS series demonstrated a tumor-to-kidney ratio substantially
in excess of 1 (range=1.56-3.32).
[0343] Uptake in other tissues was negligible with the exception of
the spleen, which showed modest, likely PSMA-mediated uptake at 4 h
p.i. followed by clearance to background levels (FIG. 8). As
expected, blood activity was significantly greater for all of the
RPS series than for .sup.177Lu-PSMA-617 (p<0.05).
.sup.177Lu-RPS-063, .sup.177Lu-RPS-061 and .sup.177Lu-RPS-068
showed the highest blood activity at 4 h p.i. (FIG. 9), while
.sup.177Lu-RPS-069, .sup.177Lu-RPS-066 and .sup.177Lu-RPS-067
showed lower blood retention at the same time point. By 24 h p.i.,
the blood activity was below 0.3% ID/g for all of the ligands, and
by 96 h it had decreased to below 0.1% ID/g. Interestingly,
although all of the RPS ligands contained the same albumin-binding
group, N.sup.6-(2-(4-iodophenyl)acetyl)-L-lysine, significant
differences (p<0.001) were observed between the shorter PEG
compounds .sup.177Lu-RPS-068 and .sup.177Lu-RPS-063 and the longer
PEG compounds .sup.177Lu-RPS-066 and .sup.177Lu-RPS-067. This
indicates that the linker influences binding to plasma proteins
and/or clearance.
[0344] The inverse correlation between PEG length and affinity for
PSMA is consistent with findings reported to date for PSMA
constructs and other targeting ligands. Small PEG linkers such as
PEG3 or PEG4 have been incorporated into small molecule drug
conjugates that target PSMA [22, 23], but constructs of this nature
to date have shown low affinity and/or poor tumor uptake. One SAR
study did establish that PEG2 and PEG4 linkers best retained PSMA
affinity in a family of PSMA-targeting contrast agents, with PEG12
and PEG24 leading to large decreases in affinity [24]. These
results were in agreement with the observation that a PEG12 linker
decreased affinity relative to PEG8 in a small molecule GCPII
ligand [25]. An SAR study of the influence of PEG linkers on
.sup.68Ga-labeled antagonists of bombesin found that affinity
slightly weakened upon each incremental extension of the PEG linker
[26]. This study also identified small differences in the
biodistribution of the ligands.
[0345] The areas under the curve (AUC) of the time-activity curves
(TACs) for tumor uptake suggest that the .sup.177Lu-labeled
RPS-061, -063, 066, -068 and -069 ligands deliver a significantly
larger dose to the tumor than does .sup.177Lu-PSMA-617 (FIG. 10).
This is confirmed by a comparison of the dose integrals in the
tumor. .sup.177Lu-RPS-068 and .sup.177Lu-RPS-063 are nearly four
times higher than .sup.177Lu-PSMA-617, while .sup.177Lu-RPS-061,
.sup.177Lu-RPS-069 and .sup.177Lu-RPS-066 are also at least two
times higher (FIG. 11).
[0346] It is likely that the tumor uptake of the .sup.177Lu-labeled
RPS ligands is also higher than other .sup.177Lu-labeled
PSMA-targeting ligands reported to date, including .sup.177Lu-PSMA
I&T, with a reported uptake in LNCaP tumors of 7.96.+-.1.76 at
1 h p.i. [27], and the recently reported .sup.177Lu-CTT1403, which
was reported to reach 46% ID/g at 72 h p.i. in PC3-PIP tumors [23].
PSMA expression is PC3-PIP tumors is higher than typically found in
human prostate cancers, notably ten-fold greater than LNCaP cells
[28], meaning that uptake of .sup.177Lu-CTT1403 is likely to be
considerably lower in LNCaP tumors. Uptake in LNCaP tumors for
.sup.177Lu-RPS-063 and .sup.177Lu-RPS-068 is on a par with the
uptake reported for .sup.131I-MIP-1095 [29], the small molecule, to
our knowledge, with the greatest uptake in LNCaP xenograft tumors
reported to date. A comparison of the TACs for .sup.177Lu-RPS-063,
.sup.177Lu-RPS-068 and .sup.131I-MIP-1095 suggests a similar AUC
for the 96 h period studied (FIG. 10).
[0347] It has previously been reported that prolonged blood
retention leads to increased tumor accumulation with time [23, 30],
a consequence presumably of an increase in the number of times the
ligand passes through the tumor bed. .sup.177Lu-RPS-068 appears to
increase from 4 h to 24 h, but this difference is not statistically
significant (p=0.26). The phenomenon is not evident among the other
trifunctional RPS ligand series after 4 h p.i. either, though it is
possible that delayed blood clearance during the first 4 h may
increase tumor uptake in this time interval. Nevertheless,
clearance of the ligands from the tumor is slow, enabling the
delivery of greater amounts of activity to the target tissue
compared to .sup.177Lu-PSMA-617. Further modification of albumin
binding by substitution of the albumin binding group may be used to
subtly modify blood clearance and reinforce the high and persistent
tumor accumulation.
[0348] In spite of promising clinical outcomes using PSMA-targeted
radioligand therapy for mCRPC, next generation ligands that (1)
overcome resistance to .beta.-particle radiation, (2) are
appropriate for treating diffuse metastatic lesions (particularly
in the bone) and (3) provide longer duration of progression free
survival are essential to continued improvements in treatment.
Although minimal toxicity is currently associated with a single
administration of .sup.177Lu-PSMA-617 or .sup.131I-MIP-1095, the
incidence of hematological toxicity and persistent xerostomia can
increase upon subsequent therapy cycles [3] while biochemical
response may decrease [3, 5]. Alpha-particle mediated therapy has
been proposed as a method of overcoming resistance to
.beta.-particles and reducing hematological toxicity [31]. Early
preclinical studies with .sup.213Bi-PSMA I&T have identified
the formation of DNA double-strand breaks in tumors in vivo [32],
while preliminary treatment of human patients with
.sup.225Ac-PSMA-617 or .sup.213Bi-PSMA-617 have led to dramatic
responses in refractory cancer [31, 33]. Nevertheless, multiple
therapy cycles were required for efficacy, leading to irreversible
xerostomia and keratoconjunctivitis sicca [33].
[0349] These early findings have demonstrated the therapeutic
potential for .alpha.-particle radiotherapy, but highlighted the
need for radioligands with greater therapeutic index that can
deliver a high dose to the tumor. Each of .sup.177Lu-RPS-061, -063,
-066, -068 and -069 shows significantly higher tumor uptake than
.sup.177Lu-PSMA-617 in LNCaP xenograft tumors, with the
corresponding increase in AUC correlating to an increase in the
dose of radioactivity delivered to the tumor. The tumor-to-kidney
ratios of .sup.177Lu-RPS-063, .sup.177Lu-RPS-068 and
.sup.177Lu-RPS-061, the three ligands with highest tumor uptake,
are 2.75.+-.0.17, 1.56.+-.0.23 and 3.64.+-.0.29, respectively, at
96 h p.i. In contrast, .sup.177Lu-CTT1403 never reaches 1.0 [23].
Although the tumor-to-kidney ratio of .sup.177Lu-PSMA-617 at the
same time point is 14.39.+-.2.2, and the ratio of tumor dose
integral to kidney dose integral over the 96 h is 1.95, these are
driven by very low kidney uptake rather than high tumor uptake. It
has been widely demonstrated that the expression of PSMA in the
kidneys of nude mice is higher than expression levels in human
kidneys [34, 35, 36], meaning that preclinical studies consistently
overestimate the dose delivered to this organ. Several
PSMA-targeted therapeutics including .sup.131I-MIP-1095 (29),
.sup.177Lu-DKFZ-617 (21) and .sup.177Lu-PSMA I&T (37) all show
early kidney concentrations at or above 100% ID/g in nude mice, yet
have been safely translated to the clinic with acceptable; albeit
not identical kidney doses.
[0350] Furthermore, additional nephroprotection schemes, including
pharmacological displacement with 2-PMPA, have been shown to reduce
activity in the kidney still further [37, 38]. Taken together,
these observations illustrate that the trifunctional RPS ligands
show both the high and persistent tumor uptake and broad
therapeutic index that are desirable for .alpha.-particle
radiotherapeutics.
SECTION 1.3 REFERENCES
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[0352] [2] Zechmann C M, Afshar-Oromieh A, Armor T, Stubbs J B,
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Haberkorn U. Radiation dosimetry and first therapy results with
a.sup.1241/.sup.131I-labeled small molecule (MIP-1095) targeting
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[0389] Section 1.4
[0390] Materials and Instrumentation.
[0391] The synthesis of RPS-074 is described below. All solvents
and reagents were purchased from commercial vendors and used
without further purification. The intermediate di-tert-butyl
(((S)-1-(tert-butoxy)-6-(3-(3-ethynylphenyl)ureido)-1-oxohexan-2-yl)carba-
moyl)-L-glutamate (406) and macropa-NCS were synthesized as
described above. Compounds were purified using silica
chromatography on VWR.RTM. High Purity Silica Gel 60 .ANG.,
preparative TLC on silica-coated glass plates (Analtech), or flash
chromatography using a CombiFlash Rf+(Teledyne Isco) system.
Preparative HPLC was performed using an XBridge.TM. Prep C18 5
.mu.m OBD.TM. 19.times.100 mm column (Waters) on a dual pump
Agilent ProStar HPLC fitted with an Agilent ProStar 325 Dual
Wavelength UV-Vis Detector. UV absorption was monitored at 220 nm
and 280 nm. A binary solvent system was used, with solvent A
comprising H.sub.2O+0.01% TFA and solvent B consisting of 90% v/v
MeCN/H.sub.2O+0.01% TFA. Purification was achieved using the
following gradient HPLC method: 0% B 0-1 min., 0-100% B 1-28 mins.,
100-0% B 28-30 mins.
[0392] Final products were identified and characterized using thin
layer chromatography, analytical HPLC and mass spectrometry. NMR
spectroscopy was used to confirm the structure of compound 406 and
macropa-NCS. NMR analyses were performed using a Bruker Avance III
500 MHz spectrometer. Spectra are reported in CDCl.sub.3 or
DMSO-d.sub.6. Analytical HPLC was performed using an XSelect.TM.
CSH.TM. C18 5 .mu.m 4.6.times.50 mm column (Waters). Mass
determinations were performed by LCMS analysis using a Waters
ACQUITY UPLC.RTM. coupled to a Waters SQ Detector 2. The purity of
all compounds evaluated in the biological assay was >95% purity
as judged by analytical HPLC.
[0393] Synthesis of Macropa-RPS-074.
##STR00069##
Preparation of tert-Butyl
N.sup.2-(1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4-aza-
hentriacontan-31-oyl)-N.sup.6-((benzyloxy)carbonyl)-L-lysinate
(402)
[0394] To a stirred mixture of Fmoc-N-amido-PEG-8-acid (663 mg, 1.0
mmol), L-N.sup..epsilon.--Z-Lys-OtBu hydrochloride (446 mg, 1.2
mmol) and HATU (456 mg, 1.2 mmol) in DMF (10 mL) was added DIPEA
(260 mg, 2.0 mmol), and the reaction was stirred overnight at room
temperature under N.sub.2. The solvent was removed under reduced
pressure and the crude residue was purified by flash chromatography
(0-10% MeOH in CH.sub.2Cl.sub.2) to give compound 2 as a colorless
oil (845 mg, 86%). Mass (ESI+): 983.0 [M+H].sup.+. Calc. Mass:
981.5.
Preparation of tert-Butyl
N.sup.2-(1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4-aza-
hentriacontan-31-oyl)-N.sup.6-(4-(4-iodophenyl)butanoyl)-L-lysinate
(403)
[0395] Compound 402 (1.45 g, 1.48 mmol) was dissolved in MeOH (25
mL). 10% Palladium on charcoal (15 mg) was added, and the
suspension was stirred in a three-neck flask at room temperature
for 10 min. The flask was evacuated and then placed under an
H.sub.2 atmosphere. The suspension was then stirred at room
temperature for 5 h before it was filtered through celite. The
filter cake was washed with MeOH, and the combined filtrate was
concentrated under reduced pressure to give the amine as a yellow
oil (1.17 g, 93%) that was used without further purification. Mass
(ESI+): 849.4 [M+H].sup.+. Calc. Mass: 848.0. To a solution of the
amine (865 mg, 1.01 mmol) and 2,5-dioxopyrrolidin-1-yl
4-(4-iodophenyl)butanoate (387 mg, 1.00 mmol) in CH.sub.2Cl.sub.2
(20 mL) was added TEA (167 .mu.L, 1.20 mmol). The resulting
solution was stirred at room temperature under Ar for 4 h. The
solution was then washed successively with 1% v/v AcOH/H.sub.2O and
brine. The organic layer was dried over MgSO.sub.4, filtered and
concentrated under reduced pressure to give a yellow oil. The crude
product was purified by flash chromatography (0-30% MeOH in
CH.sub.2Cl.sub.2) and compound 3 was isolated as a yellow oil (360
mg, 32%). Mass (ESI+): 1120.9 [M+H].sup.+. Calc. Mass: 1119.5.
Preparation of tert-Butyl
N.sup.2--((S)-10-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2,2-dimethyl-
-4,11-dioxo-3,15,18,21,24,27,30,33,36-nonaoxa-5,12-diazanonatriacontan-39--
oyl)-N.sup.6-(4-(4-iodophenyl)butanoyl)-L-lysinate (404)
[0396] A solution of 403 (360 mg, 0.32 mmol) and diethylamine (0.67
mL, 6.48 mmol) in CH.sub.2Cl.sub.2 (2 mL) was stirred at room
temperature for 7 h. The solution was concentrated under reduced
pressure and the crude residue was purified by flash chromatography
(0-30% MeOH in CH.sub.2Cl.sub.2). The fractions containing the
product were combined and concentrated to give the amine as a
yellow oil (96 mg, 33%). Mass (ESI+): 899.2 [M+H].sup.+. Calc.
Mass: 897.4. To a solution of the amine (96 mg, 107 .mu.mol) and
Fmoc-L-Lys(Boc)-OSu (62 mg, 110 .mu.mol) in CH.sub.2Cl.sub.2 (5 mL)
was added TEA (28 .mu.L, 200 .mu.mol). The mixture was stirred
overnight at room temperature under Ar. The solvent was removed
under reduced pressure and the crude product was purified by flash
chromatography (0-30% MeOH in CH.sub.2Cl.sub.2). The desired
product co-eluted with a minor impurity, therefore the mixture was
purified a second time by prep TLC (10% v/v MeOH/CH.sub.2Cl.sub.2).
Compound 404 was isolated as a colorless oil (78 mg, 51%). Mass
(ESI+): 1349.0 [M+H].sup.+. Calc. Mass: 1347.6.
Preparation of tert-Buty
N.sup.2--((S)-10-amino-2,2-dimethyl-4,11-dioxo-3,15,18,21,24,27,30,33,36--
nonaoxa-5,12-diazanonatriacontan-39-oyl)-N-(4-(4-iodophenyl)butanoyl)-L-ly-
sinate (405)
[0397] A solution of 404 (73 mg, 54 .mu.mol) and diethylamine (0.5
mL, 4.83 mmol) in CH.sub.2Cl.sub.2 (2 mL) was stirred overnight at
room temperature. The solvent was removed under reduced pressure
and the crude product was dissolved in MeOH and purified by prep
TLC (10% v/v MeOH in CH.sub.2Cl.sub.2). Amine 405 was isolated as a
pale oil (25 mg, 41%). Mass (ESI+): 1127.7 [M+H].sup.+. Calc. Mass:
1126.2.
Preparation of di-tert-butyl
(((S)-1-(tert-butoxy)-6-(3-(3-(1-(2-(2-(2-(3-((2,5-dioxopyrrolidin-1-yl)o-
xy)-3-oxopropoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)phenyl)ureido)-
-1-oxohexan-2-yl)carbamoyl)-L-glutamate (407)
[0398] A solution of 0.5M CuSO.sub.4 (100 .mu.L) and 1.5M sodium
ascorbate (100 .mu.L) was mixed until the brown color was converted
to orange. This mixture was then added to a solution of 406 (315
mg, 0.5 mmol) and azido-PEG3-NHS (177 mg, 0.5 mmol) in DMF (2 mL).
The mixture was stirred at room temperature for 2 h. It was then
diluted with CH.sub.2Cl.sub.2 and washed with H.sub.2O. The organic
layer was dried over MgSO.sub.4, filtered and concentrated under
reduced pressure to give a pale oil. The crude product was purified
by flash chromatography (0-30% MeOH in CH.sub.2Cl.sub.2) to give
compound 407 as a clear oil (460 mg, 95%). Mass (ESI+): 975.9
[M+H].sup.+. Calc. Mass: 974.5.
Preparation of di-tert-butyl
(((S)-1-(tert-butoxy)-6-(3-(3-(1-((14S,45S)-45-(tert-butoxycarbonyl)-14-(-
4-((tert-butoxycarbonyl)amino)butyl)-54-(4-iodophenyl)-12,15,43,51-tetraox-
o-3,6,9,19,22,25,28,31,34,37,40-undecaoxa-13,16,44,50-tetraazatetrapentaco-
ntyl)-1H-1,2,3-triazol-4-yl)phenyl)ureido)-1-oxohexan-2-yl)carbamoyl)-L-gl-
utamate (408)
[0399] To a solution of amine 405 (25 mg, 22 .mu.mol) in
CH.sub.2Cl.sub.2 (4 mL) was added a solution of ester 407 (24 mg,
25 .mu.mol) and TEA (7 .mu.L, 50 .mu.mol) in CH.sub.2Cl.sub.2 (1
mL). The reaction was stirred for 5 h at room temperature under Ar.
Then the reaction was concentrated under reduced pressure and the
crude residue was dissolved in EtOAc (1 mL) and purified by prep
TLC (90% EtOAc in hexanes) to give compound 408 as a pale oil (33
mg, 76%). Mass (ESI+): 994.3 [(M+2H)/2].sup.+. Calc. Mass:
1986.2.
Preparation of
(((S)-1-Carboxy-5-(3-(3-(1-((14S,45S)-45-carboxy-14-(4-(3-(2-carboxy-6-((-
16-((6-carboxypyridin-2-yl)methyl)-1,4,10,13-tetraoxa-7,16-diazacyclooctad-
ecan-7-yl)methyl)pyridin-4-yl)thioureido)butyl)-54-(4-iodophenyl)-12,15,43-
,51-tetraoxo-3,6,9,19,22,25,28,31,34,37,40-undecaoxa-13,16,44,50-tetraazat-
etrapentacontyl)-1H-1,2,3-triazol-4-yl)phenyl)ureido)pentyl)carbamoyl)-L-g-
lutamic Acid (macropa-RPS-074)
##STR00070##
[0401] Compound 408 (33 mg, 16 .mu.mol) was dissolved in
CH.sub.2Cl.sub.2 (2 mL). Then TFA (0.5 mL) was added and the
reaction was stirred overnight at room temperature. The solvent was
removed under N.sub.2 flow and the crude product was lyophilized to
give a white residue (22 mg, 83%). Mass (ESI+): 832.0
[(M+2H)/2].sup.+. Calc. Mass: 1661.8. To a solution of the free
amine (13 mg, 7.8 .mu.mol) and TEA (0.22 mL, 1.56 mmol) in DMF (1
mL) was added a solution of macropa-NCS (6 mg, 10 .mu.mol) in DMF
(1 mL). The resulting mixture was stirred for 90 min at room
temperature. The reaction was concentrated under reduced pressure
and the crude product was purified by prep HPLC. The peak
corresponding to the desired product was collected and lyophilized
to give macropa-RPS-074 as a white powder (4.5 mg, 26%). Mass
(ESI+): 1126.6 [(M+2H)/2].sup.+. Calc. Mass: 2251.3.
[0402] Synthesis of DOTA-Lys-IPBA
##STR00071##
Preparation of 2,5-dioxopyrrolidin-1-yl 4-(4-iodophenyl)butanoate
(409)
[0403] A solution of 4-(4-iodophenyl)butanoic acid (1.16 g, 4.0
mmol), N-hydroxysuccinimide (483 mg, 4.2 mmol), EDC.HCl (768 mg,
4.0 mmol) and 4-DMAP (5.8 mg, 47 .mu.mol) in CH.sub.2Cl.sub.2 (30
mL) was stirred for 20 h. Then the reaction mixture was washed
successively with 1M HCl, saturated NaHCO.sub.3 and brine. The
organic layer was dried over MgSO.sub.4, filtered and concentrated
under reduced pressure to give NHS ester 409 as a white powder
(1.29 g, 83%). .sup.1H NMR (CDCl.sub.3, 500 MHz): .delta. 7.61 (d,
2H, J=7.2 Hz). 6.95 (d, 2H, J=7.6 Hz), 2.83 (s, 4H), 2.67 (t, 2H,
J=7.6 Hz), 2.59 (t, 2H, J=7.3 Hz), 2.03 (quint, 2H, J=7.3 Hz).
Preparation of
N.sup.2-(tert-butoxycarbonyl)-N.sup.6-(4-(4-iodophenyl)butanoyl)-L-lysine
(410)
[0404] Boc-L-Lys-OH (871 mg, 3.53 mmol) was suspended in DMF (10
mL) and stirred at room temperature. To the stirred suspension was
slowly added a solution of NHS ester 409 (1.29 g, 3.33 mmol) and
NEt.sub.3 (557 .mu.L, 4.00 mmol) in DMF (5 mL). The resulting
suspension was stirred overnight at room temperature. The reaction
was quenched with 1M HCl (2 mL), and the solvent was removed under
reduced pressure. The crude residue was dissolved in
CH.sub.2Cl.sub.2 and washed successively with 1M HCl, saturated
NaHCO.sub.3 solution and brine. The organic fraction was dried over
MgSO.sub.4, filtered and concentrated under reduced pressure to
give Boc-Lys-IPBA (410) as a clear foam (1.25 g, 72%). .sup.1H NMR
(CDCl.sub.3, 500 MHz): .delta. 7.57 (d, 2H, J=7.7 Hz), 6.91 (d, 2H,
J=7.8 Hz), 5.94 (br s, 1H), 5.32 (br s, 1H), 4.21 (m, 1H), 3.21 (m,
2H), 2.56 (t, 2H, J=7.6 Hz), 2.15 (t, 2H, J=7.1 Hz), 1.90 (quint,
2H, J=7.5 Hz), 1.88 (m, 1H), 1.69 (m, 1H), 1.51 (m, 2H), 1.42 (s,
9H), 1.41 (m, 2H). Mass (ESI+): 519.3 (M+H)+. Calc. Mass:
518.4.
Preparation of N.sup.6-(4-(4-iodophenyl)butanoyl)-L-lysine
(411)
[0405] Boc-Lys-IPBA (518 mg, 1.0 mmol) was dissolved in 10 mL of a
20% v/v TFA/CH.sub.2Cl.sub.2 solution and stirred overnight at room
temperature. The solvents were removed under a stream of N.sub.2
and Lys-IPBA (411) was isolated as a colorless oil (402 mg; 96%).
.sup.1H NMR (DMSO, 500 MHz): .delta. 7.75 (br s, 1H), 7.61 (d, 2H,
J=7.8 Hz), 6.99 (d, 2H, J=7.8 Hz), 3.79 (m, 1H), 2.99 (m, 2H), 2.02
(t, 2H, J=7.3 Hz), 1.74 (quint, 2H, J=7.4 Hz), 1.37 (m, 4H), 1.24
(m, 2H). Mass (ESI+): 419.2 (M+H)+. Calc. Mass: 418.3.
Preparation of
2,2',2'',2'''-(2-(4-(3-((S)-1-carboxy-5-(4-(4-iodophenyl)butanamido)penty-
l)thioureido)benzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetra-
acetic Acid (DOTA-Lys-IPBA)
[0406] To a solution of Lys-IPBA (11 mg, 26 .mu.mol) and DIPEA (17
.mu.L, 100 .mu.mol) in DMF (1 mL) was added a solution of
p-SCN-Bn-DOTA.2.5Cl.2.5H.sub.2O (8 mg, 11.6 .mu.mol) in H.sub.2O (1
mL). The reaction was stirred at room temperature for 4 h. The
solvent was removed under reduced pressure and the crude residue
was purified by prep HPLC. The peak corresponding to the product
was collected and lyophilized to give DOTA-Lys-IPBA as a white
powder (5 mg, 43%). .sup.1H NMR (DMSO, 500 MHz): .delta. 9.72 (br
s, 1H), 7.89 (d, 2H, J=7.6 Hz), 7.77 (m, 1H), 7.61 (d, 2H, J=7.1
Hz), 7.51 (d, 2H, J=7.8 Hz), 7.24 (m, 2H), 6.99 (d, 2H, J=7.8 Hz),
4.84 (m, 1H), 3.70-3.04 (m, 14H), 3.01 (m, 4H), 2.02 (t, 2H, J=7.1
Hz), 1.76 (m, 4H), 1.39 (m, 2H), 1.31 (m, 2H). Mass (ESI+): 971.0
(M+H)+. Calc. Mass: 969.9.
[0407] Radiochemistry.
[0408] All reagents were purchased from Sigma Aldrich unless
otherwise noted, and were reagent grade. Hydrochloric acid (HCl)
was traceSELECT.RTM. (>99.999%) for trace analysis quality.
Aluminum-backed silica thin layer chromatography (TLC) plates were
purchased from Sigma Aldrich. Stock solutions of 0.05M HCl and 1M
NH.sub.4OAc were prepared by dilution in Milli-Q.RTM. water.
[0409] .sup.225Ac-RPS-074:
[0410] To a solution of .sup.225Ac(NO.sub.3).sub.3 (Oak Ridge
National Laboratory, USA) in 0.05M HCl (16.7-21.0 MBq in 950 .mu.L)
was added 20 .mu.L of a 1 mg/mL solution of RPS-074 in DMSO. The pH
was increased to 5-5.5 by addition of 90 .mu.L M NH.sub.4OAc. The
reaction was gently shaken for 20 min at 25.degree. C. on an
Eppendorf Thermomixer.RTM. C (VWR). Then the reaction was diluted
with H.sub.2O (9 mL) and passed through a pre-activated Sep-Pak C18
Light cartridge (Waters). The reaction vial and cartridge were
washed with H.sub.2O (5 mL) and the product was eluted with 500
.mu.L of EtOH followed by 500 .mu.L normal saline (0.9% NaCl in
deionized H.sub.2O; VWR). The eluate was diluted to 4 mL in normal
saline to give a stock solution with a radioactivity concentration
of 1.1-1.5 MBq/mL. An aliquot was removed from the final solution
and spotted onto an aluminum-backed silica TLC plate to determine
radiochemical impurity. An aliquot of the
.sup.225Ac(NO.sub.3).sub.3 solution in 0.05M HCl was spotted in a
parallel lane as a control. The plate was immediately run in a 10%
v/v MeOH/10 mM EDTA mobile phase, and then allowed to stand for 8 h
to enable radiochemical equilibrium to be reached. The plate was
visualized on a Cyclone Plus Storage Phosphor System (Perkin Elmer)
following a 3 min exposure on the phosphor screen. The
radiochemical purity was expressed as a ratio of .sup.225Ac-RPS-074
to total activity and was determined to be 98.1%. The plate was
visualized again 16 h later to confirm purity.
[0411] .sup.225Ac-DOTA-Lys-IPBA:
[0412] To a solution of .sup.225Ac(NO.sub.3).sub.3 (Oak Ridge
National Laboratory, USA) in 0.05M HCl (5.0 MBq in 900 .mu.L) was
added 30 .mu.L of a 1 mg/mL solution of DOTA-Lys-IPBA in DMSO. The
pH was increased to 5-5.5 by addition of 80 .mu.L 1M NH.sub.4OAc,
and the reaction was heated for 25 min at 95.degree. C. on an
Eppendorf Thermomixer.RTM. C (VWR). Then the reaction mixture was
diluted with H.sub.2O (9 mL) and passed through a pre-activated
Sep-Pak C18 Light cartridge (Waters). The reaction vial and
cartridge were washed with H.sub.2O (5 mL) and the product was
eluted with 200 .mu.L of a 50% v/v EtOH/saline solution followed by
800 .mu.L normal saline (0.9% NaCl in deionized H.sub.2O; VWR).
Radiochemical purity (96%) was determined by radioTLC as described
above.
[0413] Cell Culture. The PSMA expressing human prostate cancer cell
line, LNCaP, was obtained from the American Type Culture
Collection. Cell culture supplies were obtained from Invitrogen
unless otherwise noted. LNCaP cells were maintained in RPMI-1640
medium supplemented with 10% fetal bovine serum (Hyclone), 4 mM
L-glutamine, 1 mM sodium pyruvate, 10 mM
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 2.5
mg/mL D-glucose, and 50 .mu.g/mL gentamicin in a humidified
incubator at 37.degree. C./5% CO.sub.2. Cells were removed from
flasks for passage or for transfer to 12-well assay plates by
incubating them with 0.25% trypsin/ethylenediaminetetraacetic acid
(EDTA).
[0414] In Vitro Determination of IC.sub.50.
[0415] IC.sub.50 values of the non-labeled, metal-free ligands were
determined by screening in a multi-concentration competitive
binding assay against
.sup.99mTc-((7S,12S,16S)-1-(1-(carboxymethyl)-1H-imidazol-2-yl)-2-((1-(ca-
rboxymethyl)-1H-imidazol-2-yl)methyl)-9,14-dioxo-2,8,13,15-tetraazaoctadec-
ane-7,12,16,18-tetracarboxylic acid technetium tricarbonyl complex)
(.sup.99mTc-MIP-1427), with K.sub.d=0.64.+-.0.46 nM [1] for binding
to PSMA on LNCaP cells, according to previously described methods
[2] with small modifications. Briefly, LNCaP cells were plated 72 h
prior to the experiment to achieve a density of approximately
5.times.10.sup.5 cells/well (in triplicate) in RPMI-1640 medium
supplemented with 0.25% bovine serum albumin. The cells were
incubated for 2 h with 1 nM .sup.99mTc-MIP-1427 in RPMI-1640 medium
containing 0.00125% w/v bovine serum albumin [3] in the presence of
0.001-10,000 nM test compounds. Radioactive incubation media was
then removed by pipette and the cells were washed twice using 1 mL
ice-cold PBS 1.times. solution. Cells were harvested from the
plates following treatment with 1 mL 1M NaOH and transferred to
tubes for radioactive counting using a 2470 Wizard.sup.2 Automatic
Gamma Counter (Perkin Elmer). Standard solutions (10% of activity
added to each well) were prepared to enable decay correction.
Cell-specific activity was corrected for non-specific binding of
.sup.99mTc-MIP-1427. IC.sub.50 values were determined by fitting
the data points to a sigmoidal Hills1 curve in Origin software.
[0416] Inoculation of mice with xenografts. All animal studies were
approved by the Institutional Animal Care and Use Committee of
Weill Cornell Medicine and were undertaken in accordance with the
guidelines set forth by the USPHS Policy on Humane Care and Use of
Laboratory Animals. Animals were housed under standard conditions
in approved facilities with 12 h light/dark cycles. Food and water
was provided ad libitum throughout the course of the studies. Male
BALB/c athymic nu/nu mice were purchased from the Jackson
Laboratory. For inoculation in mice, LNCaP cells were suspended at
4.times.10.sup.7 cells/mL in a 1:1 mixture of PBS:Matrigel (BD
Biosciences). Each mouse was injected in the left flank with 0.25
mL of the cell suspension. Biodistributions were conducted when
tumors were in the range 200-800 mm.sup.3, while therapy studies
were initiated when tumors were in the range 50-900 mm.sup.3.
[0417] Biodistribution Studies in LNCaP Xenograft Mice.
[0418] LNCaP xenograft tumor-bearing mice (4 per time point per
compound) were injected intravenously with a bolus injection of 105
kBq and 320 ng (142 .mu.mol) of .sup.225Ac-RPS-074. The mice were
sacrificed at 4 h, 24 h, 7 d, 14 d and 21 d post injection. A blood
sample was removed, and a full biodistribution study was conducted
on the following organs (with contents): heart, lungs, liver, small
intestine, large intestine, stomach, spleen, pancreas, kidneys,
muscle, bone and tumor. Tissues were weighed and counted on a 2470
Wizard Automatic Gamma Counter (Perkin Elmer). Counts were
corrected for decay and for activity injected, and tissue uptake
was expressed as percent injected dose per gram (% ID/g). Standard
error measurement was calculated for each data point.
[0419] Therapy Study in LNCaP Xenograft Mice.
[0420] LNCaP xenograft tumor-bearing mice were randomly assigned to
5 groups (7 mice per group). One group was injected intravenously
with a bolus injection of 148 kBq and 93 ng (41 .mu.mol)
.sup.225Ac-RPS-074. The second treatment group was injected with 74
kBq and 47 ng (21 .mu.mol) .sup.225Ac-RPS-074. The third treatment
group was injected with 37 kBq and 23 ng (10 .mu.mol)
.sup.225Ac-RPS-074. The fourth group was injected with the same
volume of vehicle. The fifth group was injected with 133 kBq
.sup.225Ac-DOTA-Lys-IPBA. Tumor dimensions were measured and
recorded three times weekly with digital calipers, and tumor
volumes were calculated using the modified ellipsoid equation,
V=0.5*length*width*width [4]. Mice were sacrificed after tumors
reached 2000 mm.sup.3 or if they showed any visible signs of
distress, including loss of body weight, lack of appetite,
excessive lethargy or formation of sores and rashes. Body weight
was measured with a digital balance twice weekly, and mice were
monitored for signs of distress. The mice were photographed weekly
to visually confirm changes in tumor volume.
[0421] Imaging of Treated Mice by .mu.PET/CT.
[0422] .sup.68Ga-PSMA-11 (also known as .sup.68Ga-HBED-CC) was
prepared as previously reported [5]. Eight mice were injected
intravenously with 5.5 MBq .sup.68Ga-PSMA-11, 75 days after
injection of either 138 kBq or 74 kBq .sup.225Ac-RPS-074. The mice
were imaged using PET/CT (Inveon.TM.; Siemens Medical Solutions,
Inc.) at 1 h post-injection following inhalation anesthetization
with isoflurane. Total acquisition time was 30 min. A CT scan was
obtained immediately before the acquisition for both anatomical
co-registration and attenuation correction. Images were
reconstructed using the Inveon.TM. software supplied by the
vendor.
[0423] In Vitro and In Vivo Evaluation of RPS-074
[0424] The IC.sub.50 value of RPS-074 was determined in vitro using
a multi-concentration competitive binding assay against
.sup.99mTc-MIP-1427, which displays affinity for PSMA on LNCaP
cells. It was demonstrated that the IC.sub.50 of RPS-074 was
12.0.+-.3.4 nM, a value that is consistent with the reported PSMA
affinities of structurally analogous trifunctional ligands [6]. The
biodistribution of RPS-074 was examined in vivo in LNCaP xenograft
tumor-bearing mice. Mice were injected intravenously with a bolus
injection of 105 kBq and 320 ng (142 .mu.mol) of
.sup.225Ac-RPS-074. The mice were sacrificed at 4 h, 24 h, 7 d, 14
d and 21 d post injection. FIG. 12 demonstrates that uptake of
.sup.225Ac-RPS-074 was evident in the blood (12.3.+-.0.5% ID/g),
the lungs (5.0.+-.0.2% ID/g), the kidneys (6.7.+-.0.4% ID/g) and
the tumor (5.8.+-.0.3% ID/g) at 4 h post injection (p.i.). By 24 h
p.i., the activity in non-target tissue, including kidneys
(3.0.+-.0.3% ID/g), cleared in concert with blood clearance, while
activity in the tumor increased to 12.7.+-.1.5% ID/g (FIG. 12). By
7 d p.i., activity in the tumor remained high (9.5.+-.1.5% ID/g),
while the activity in the blood and every other tissue was less
than 1% ID/g (FIG. 12). Persistent tumor uptake (11.9.+-.1.5% ID/g)
was evident at 14 d p.i., with all other tissues becoming largely
indistinguishable from background. By 21 d p.i., an anti-tumor
effect was evident, with only 1 mouse still bearing a tumor.
Notwithstanding the absence of tumors, activity in the non-target
tissue remained indistinguishable from background.
[0425] .sup.225Ac-RPS-074 showed excellent complex stability over 3
weeks even when tumors were absent. Biodistribution studies
demonstrated that no accumulation of signal was evident in the
liver or bone, two organs that typically take up free
.sup.225Ac.sup.3+ [7]. .sup.225Ac-RPS-074 also demonstrated a
favorable pharmacokinetic profile; the tumor-to-kidney and
tumor-to-blood ratios rapidly favor the tumor. By 24 h p.i., the
tumor-to-kidney ratio reached 4.3.+-.0.7 while at 7 d and 14 d p.i.
it is 15.0.+-.2.9 and 62.2.+-.9.5, respectively. The tumor-to-blood
ratio at the same time points is 3.3.+-.0.5, 137.5.+-.30.4 and
995.8.+-.139.7. Significant differences in the pharmacokinetic
profile demonstrates that the dose absorbed by each tissue will be
different.
[0426] Therapeutic Evaluation in LNCaP Xenograft Mice
[0427] LNCaP xenograft were randomly assigned to 5 groups and
treated with a bolus injection of 148 kBq and 93 ng (41
pmol).sup.225Ac-RPS-074, 74 kBq and 47 ng (21 pmol)
.sup.225Ac-RPS-074, 37 kBq and 23 ng (10 pmol).sup.225Ac-RPS-074,
133 kBq .sup.225Ac-DOTA-Lys-IPBA, or vehicle control. A significant
antitumor effect was observed in the mice treated with 138 kBq and
74 kBq of .sup.225Ac-RPS-074. In the 138 kBq treatment group, 6/7
(86%) of tumors were not detectable (<0.5 mm.sup.3) at 75 d post
injection, while 1/7 (14%) of tumors were not detectable in the 74
kBq group. The distribution of initial tumor volumes was 100-624
mm.sup.3 and 64-455 mm.sup.3 for the two groups, respectively (FIG.
13). Tumor volume decreased in the 74 kBq group for as much as 42 d
post injection before 6/7 (86%) of tumor volumes began to increase
again. The absence of tumors was confirmed by .mu.PET/CT imaging
with .sup.68Ga-PSMA-11 (FIG. 14) prior to the collection of samples
for pathology. Those tumors that re-emerged in the 74 kBq treatment
group were shown by imaging to express PSMA. Physiologic uptake was
also evident in the kidneys and salivary glands.
[0428] FIG. 13 demonstrates that both the 37 kBq treatment group
and the positive control group, which received 133 kBq
.sup.225Ac-DOTA-Lys-IPBA, showed an initial effect relative to the
vehicle group, but tumor volumes increased from a starting volume
of 99-331 mm.sup.3 and 233-859 mm.sup.3, respectively, to a final
volume of greater than 2000 mm.sup.3. A clear dose-response was
evident in this study. Up to 42 days p.i., the 74 kBq and 138 kBq
treatment groups behaved similarly, but while the tumor volume of
5/7 (71%) of mice in the former group was measured to be less than
1 mm.sup.3, the tumors progressively returned. In contrast, the
tumors of the mice in the 37 kBq treatment group appeared to grow
at a similar rate to the untreated tumors.
[0429] Every mouse in the 138 kBq treatment group survived the 75 d
study (FIG. 15). In contrast, in each of the other groups at least
one mouse was sacrificed prior to the termination of the study due
to excessive tumor growth. The survival curves for the 37 kBq group
and the 133 kBq .sup.225Ac-DOTA-Lys-IPBA positive control group are
similar, with 100% of mice surviving the first 21 d. In contrast,
only 1/7 (14%) of the untreated mice survived to this time point.
No toxic effects were visible in any of the groups. The variation
in body weight during the 75 day study was 92-106% of the original
measurement. The remaining mice were sacrificed at 75 d post
injection and the tumor (if present), kidneys, liver, parotid
glands and sublingual glands were excised and examined for evidence
of damage.
[0430] Further Exemplary Compounds of the Present Technology
[0431] The following compounds of the present technology were
synthesized and characterized via similar protocols and methods as
described above.
##STR00072## ##STR00073## ##STR00074## ##STR00075## ##STR00076##
##STR00077## ##STR00078## ##STR00079##
SECTION 1.4 REFERENCES
[0432] [1] Hillier S M, Maresca K P, Lu G, Merkin R D, Marquis J C,
Zimmerman C N, Eckelman W C, Joyal J L, Babich J W.
.sup.99mTc-Labeled Small-Molecule Inhibitors of Prostate-Specific
Membrane Antigen for Molecular Imaging of Prostate Cancer. J Nucl
Med. 2013; 54:1369-76. [0433] [2] Kelly J M, Amor-Coarasa A,
Nikolopoulou A, Wustemann T, Barelli P, Kim D, Williams C. Jr,
Zheng X, Bi C, Hu B, Warren J D, Hage D S, DiMagno S G, Babich J W.
Dual-Target Binding Ligands with Modulated Pharmacokinetics for
Endoradiotherapy of Prostate Cancer. J Nucl Med. 2017;
58:1442-1449. [0434] [3] Benesovi M, Umbricht C A, Schibli R, Mller
C. Albumin-Binding PSMA Ligands: Optimization of the Tissue
Distribution Profile. Mol Pharm. 2018; 15:934-946. [0435] [4]
Jensen M M, Jorgensen J T, Binderup T, Kjor A. Tumor volume in
subcutaneous mouse xenografts measured by microCT is more accurate
and reproducible than determined by .sup.18F-FDG-microPET or
external caliper. BMC Med Imaging 2008; 8:16. [0436] [5]
Amor-Coarasa A, Kelly J M, Gruca M, Nikolopoulou A, Vallabhajosula
S, Babich J W. Continuation of comprehensive quality control of the
itG .sup.68Ge/.sup.68Ga generator and production of
.sup.68Ga-DOTATOC and .sup.68Ga-PSMA-HBED-C C for clinical research
studies. Nucl Med Biol. 2017; 53:37-39. [0437] [6] Kelly J,
Amor-Coarasa A, Ponnala S, Nikolopoulou A, Williams C., Jr, Schlyer
D, Zhao Y, Kim D, Babich J W. Trifunctional PSMA-Targeting
Constructs for Prostate Cancer with Unprecedented Localization to
LNCaP Tumors. Eur J Nucl Med Mol Imaging 2018; In press. [0438] [7]
Miederer M, Scheinberg D A, McDevitt M R. Realizing the potential
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alpha-particle therapy applications. Adv Drug Deliv Rev. 2008;
60:1371-1382.
[0439] While certain embodiments have been illustrated and
described, a person with ordinary skill in the art, after reading
the foregoing specification, can effect changes, substitutions of
equivalents and other types of alterations to the compounds of the
present technology or salts, pharmaceutical compositions,
derivatives, prodrugs, metabolites, tautomers or racemic mixtures
thereof as set forth herein. Each aspect and embodiment described
above can also have included or incorporated therewith such
variations or aspects as disclosed in regard to any or all of the
other aspects and embodiments.
[0440] The present technology is also not to be limited in terms of
the particular aspects described herein, which are intended as
single illustrations of individual aspects of the present
technology. Many modifications and variations of this present
technology can be made without departing from its spirit and scope,
as will be apparent to those skilled in the art. Functionally
equivalent methods within the scope of the present technology, in
addition to those enumerated herein, will be apparent to those
skilled in the art from the foregoing descriptions. Such
modifications and variations are intended to fall within the scope
of the appended claims. It is to be understood that this present
technology is not limited to particular methods, reagents,
compounds, compositions, labeled compounds or biological systems,
which can, of course, vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
aspects only, and is not intended to be limiting. Thus, it is
intended that the specification be considered as exemplary only
with the breadth, scope and spirit of the present technology
indicated only by the appended claims, definitions therein and any
equivalents thereof.
[0441] The embodiments, illustratively described herein may
suitably be practiced in the absence of any element or elements,
limitation or limitations, not specifically disclosed herein. Thus,
for example, the terms "comprising," "including," "containing,"
etc. shall be read expansively and without limitation.
Additionally, the terms and expressions employed herein have been
used as terms of description and not of limitation, and there is no
intention in the use of such terms and expressions of excluding any
equivalents of the features shown and described or portions
thereof, but it is recognized that various modifications are
possible within the scope of the claimed technology. Additionally,
the phrase "consisting essentially of" will be understood to
include those elements specifically recited and those additional
elements that do not materially affect the basic and novel
characteristics of the claimed technology. The phrase "consisting
of" excludes any element not specified.
[0442] In addition, where features or aspects of the disclosure are
described in terms of Markush groups, those skilled in the art will
recognize that the disclosure is also thereby described in terms of
any individual member or subgroup of members of the Markush group.
Each of the narrower species and subgeneric groupings falling
within the generic disclosure also form part of the invention. This
includes the generic description of the invention with a proviso or
negative limitation removing any subject matter from the genus,
regardless of whether or not the excised material is specifically
recited herein.
[0443] As will be understood by one skilled in the art, for any and
all purposes, particularly in terms of providing a written
description, all ranges disclosed herein also encompass any and all
possible subranges and combinations of subranges thereof. Any
listed range can be easily recognized as sufficiently describing
and enabling the same range being broken down into at least equal
halves, thirds, quarters, fifths, tenths, etc. As a non-limiting
example, each range discussed herein can be readily broken down
into a lower third, middle third and upper third, etc. As will also
be understood by one skilled in the art all language such as "up
to," "at least," "greater than," "less than," and the like, include
the number recited and refer to ranges which can be subsequently
broken down into subranges as discussed above. Finally, as will be
understood by one skilled in the art, a range includes each
individual member.
[0444] All publications, patent applications, issued patents, and
other documents (for example, journals, articles and/or textbooks)
referred to in this specification are herein incorporated by
reference as if each individual publication, patent application,
issued patent, or other document was specifically and individually
indicated to be incorporated by reference in its entirety.
Definitions that are contained in text incorporated by reference
are excluded to the extent that they contradict definitions in this
disclosure.
[0445] Other embodiments are set forth in the following claims,
along with the full scope of equivalents to which such claims are
entitled.
* * * * *
References