U.S. patent application number 17/520603 was filed with the patent office on 2022-03-24 for inhibitors of protein arginine deiminases (pads) and methods of preparation and use thereof.
The applicant listed for this patent is University of Massachusetts. Invention is credited to Aaron Muth, Venkataraman Subramanian, Paul R. Thompson.
Application Number | 20220089549 17/520603 |
Document ID | / |
Family ID | |
Filed Date | 2022-03-24 |
United States Patent
Application |
20220089549 |
Kind Code |
A1 |
Thompson; Paul R. ; et
al. |
March 24, 2022 |
INHIBITORS OF PROTEIN ARGININE DEIMINASES (PADS) AND METHODS OF
PREPARATION AND USE THEREOF
Abstract
The invention provides novel inhibitors or inactivators of
protein arginine deiminases, pharmaceutical compositions and
methods of use thereof. The invention also relates to molecular
probes based on such compounds and methods of use thereof.
Inventors: |
Thompson; Paul R.;
(Wellesley, MA) ; Muth; Aaron; (Brighton, MA)
; Subramanian; Venkataraman; (Worcester, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Massachusetts |
Boston |
MA |
US |
|
|
Appl. No.: |
17/520603 |
Filed: |
November 5, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16464317 |
May 28, 2019 |
11208386 |
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PCT/US17/63324 |
Nov 27, 2017 |
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17520603 |
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62429099 |
Dec 2, 2016 |
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International
Class: |
C07D 235/14 20060101
C07D235/14; C07D 403/12 20060101 C07D403/12; C12Q 1/34 20060101
C12Q001/34; G01N 33/58 20060101 G01N033/58 |
Goverment Interests
STATEMENT REGARDING FEDERALLY FUNDED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under Grant
No. GM079357 awarded by the National Institutes of Health. The
Government has certain rights in the invention.
Claims
1-37. (canceled)
38. A molecular imaging probe having the structural formula:
A.sub.F-L.sub.F-W (II) wherein A.sub.F is a group comprising an
optically detectable moiety; L.sub.F is a linking group; and W is
group comprising a benzimidazole moiety, or a derivative or analog
thereof, capable of inhibiting or inactivating a biological
function of a protein arginine deiminase.
39. The molecular imaging probe of claim 38, wherein A.sub.F is a
group comprising a fluorophore.
40. The molecular imaging probe of claim 39, wherein the
fluorophore is selected from the group consisting of xanthene dyes,
cyanine dyes, coumarin dyes and bodipy dyes.
41. The molecular imaging probe of claim 40, wherein the
fluorophore is a xanthene dye selected from the group consisting of
fluorescein, eosins, and rhodamines.
42. The molecular imaging probe of claim 40 wherein the fluorophore
is a cyanine dye.
43. The molecular imaging probe of claim 40, wherein the
fluorophore is a coumarin dye.
44. The molecular imaging probe of claim 40, wherein the
fluorophore is a bodipy dye.
45. The molecular imaging probe of claim 38, wherein the protein
arginine deiminase is selected from the group consisting of: PAD1,
PAD2, PAD3 and PAD4.
46. The molecular imaging probe of claim 45, wherein the protein
arginine deiminase is PAD2.
47. The molecular imaging probe of claim 45, wherein the protein
arginine deiminase is PAD4.
48. The molecular imaging probe of claim 38, wherein W is a
monovalent radical derived from a compound having the structural
formula: ##STR00142## wherein, each of R.sub.a and R.sub.b is
independently selected from the group consisting of H, D and F; L
is a bivalent hydrocarbyl linker, optionally with one or more
carbon atoms replaced by a heteroatom selected from the group
consisting of O, S and N; X is a halogen atom; Y is N, O or S;
provided that when Y is S or O, its bonding to the adjacent carbons
are single bonds; Z is N--R.sub.1, O or S; R.sub.1 is selected from
the group consisting of: H, a C.sub.1-6 alkyl, OH, a C.sub.1-3
alkoxy, CF.sub.3, COCH.sub.3, and COCF.sub.3 groups; each of
R.sub.2, R.sub.3, R.sub.4 and R.sub.5 is independently selected
from the group consisting of: H, hydroxyl, halogen atom, C.sub.1-6
alkyl, C.sub.1-6 alkoxy, alkynyl, CF.sub.2R.sub.c and
OCF.sub.2R.sub.c groups, where R.sub.c is H, F or alkyl; and
R.sub.6 is a group comprising a cyclic alkyl or aryl moiety.
49. A method for identifying a protein arginine deiminase inhibitor
or inactivator, comprising: performing a competitive assay wherein
a test compound competes with a molecular imaging probe according
to claim 38 to bind to a protein arginine deiminase; and measuring
fluorescence to determine an amount of fluorescent protein arginine
deiminase present in the test assay.
50. The method of claim 49, further comprising: performing a
control assay wherein the molecular imaging probe binds to the
protein arginine deiminase; and measuring fluorescence to determine
an amount of fluorescent protein arginine deiminase present in the
control assay.
51. The method of claim 50, wherein a change in fluorescence in the
assay greater than a pre-selected value when compared to the
control assay is indicative that the test compound is an inhibitor
to the protein arginine deiminase.
52. The method of claim 50, wherein the change in fluorescence in
the assay is a decrease in fluorescence in the assay.
Description
PRIORITY CLAIMS AND RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 62/429,099, filed Dec. 2, 2016, the entire content
of which is incorporated herein by reference for all purposes.
TECHNICAL FIELDS OF THE INVENTION
[0003] The invention generally relates to therapeutic compounds,
pharmaceutical compositions and methods thereof. More particularly,
the invention provides inhibitors or inactivators of protein
arginine deiminases, pharmaceutical compositions and methods of
preparation and use thereof. The invention also relates to
molecular probes based on such compounds and methods of preparation
and use thereof.
BACKGROUND OF THE INVENTION
[0004] A significant portion of the general population is affected
by immune system disorders, which include abnormally low activity
or over activity of the immune system. In cases of autoimmune
diseases, the body's overly active immune system attacks and
damages its own tissues. In cases of an overly inactive immune
system, the body's ability to fight invaders decreases due to
immune deficiency, leaving the patient vulnerable to infections.
Currently, treatment for autoimmune diseases focuses on relieving
symptoms because there is no curative therapy.
[0005] Immune system disorders, such as rheumatoid arthritis (RA),
multiple sclerosis, lupus, and ulcerative colitis, are placing an
increasing burden on society, impairing the health and lives of
those affected. Although medications have been developed to treat
some of these diseases and conditions, the available treatments are
often limited in terms of clinical effectiveness and at the same
time have undesirable side effects.
[0006] Protein arginine deiminases (PADs) are a unique family of
enzymes that catalyzes a form of post-translational modification
called arginine deimination or citrullination: the hydrolysis of
peptidyl-arginine to form peptidyl-citrulline on histones,
fibrinogen, and other biologically relevant proteins. The
post-translational modification of histones has significant effects
on overall chromatin function.
[0007] The PAD reaction involves the hydrolysis of the guanidinium
group of arginine to generate citrullinated proteins. This reaction
is a calcium-dependent process wherein calcium binding triggers a
conformational change that moves a nucleophilic cysteine residue
into the active site, resulting in a >10,000-fold increase in
PAD activity. Overexpression and/or increased PAD activity is
observed in several diseases, including rheumatoid arthritis,
Alzheimer's disease, multiple sclerosis, lupus, Parkinson's
disease, and cancer. (Liu, et al. 2011 PLoS One 6, e21314; Kearney,
et al. 2005 Biochemistry 44, 10570-10582.)
[0008] There are five PAD isozymes (PADs 1-4 and 6) with unique
cellular and tissue distribution patterns, where only PADs 1-4 have
been isolated in their catalytically active form. The PADs are
uniquely distributed both within the cell and throughout the body.
Specifically, all the PADs can be found in the cellular cytoplasm
while only PADs 2, 3 and 4 are expressed in the nucleus. (Jones, et
al. 2009 Curr Opin Drug Discov Devel 12, 616-627. Vossenaar, et al.
2003 Bioessays 25, 1106-1118; Stone, et al. 2005 Biochemistry 44,
13744-13752; Fuhrmann, et al. 2015 Chem Rev 115, 5413-5461;
Nakashima, et al. 2002 J Biol Chem 277, 49562-49568; Cherrington,
et al. 2010 PLoS One 5, el 1768; Jang, et al. 2011 J Neuropathol
Exp Neurol 70, 116-124; Li, et al. 2016 PLoS One 11, e0147503.)
[0009] There is an urgent and growing need for novel therapeutics
and treatment methods that provide improved clinical effectiveness
with reduced side effects, in particular through safe and effective
inhibition or inactivation of PADs.
SUMMARY OF THE INVENTION
[0010] The invention provides novel inhibitors or inactivators of
PADs, pharmaceutical compositions and methods of preparation and
use thereof. The compounds and pharmaceutical compositions of the
invention may be used to treat immune system disorders and
inflammatory diseases and conditions (e.g., rheumatoid arthritis,
lupus).
[0011] The invention also provides novel molecular probes (e.g.,
imaging probes) for PADs based on the inhibitors or inactivators of
PADs disclosed herein in conjugation with detectable labels such as
fluorescent dyes, and methods of preparation and use thereof. The
molecular imaging probes of the invention may be used to screen or
identify compounds for PAD inhibition or inactivation.
[0012] In one aspect, the invention generally relates to a compound
having the structural formula
##STR00001##
wherein
[0013] each of R.sub.a and R.sub.b is independently selected from
the group consisting of H, D and F;
[0014] L is a bivalent hydrocarbyl linker, optionally with one or
more carbon atoms replaced by a heteroatom selected from the group
consisting of O, S and N;
[0015] X is a halogen atom;
[0016] Y is N, O or S; provided that when Y is S or O, its bonding
to the adjacent carbons are single bonds;
[0017] Z is N--R.sub.1, O or S;
[0018] R.sub.1 is selected from the group consisting of: H, a
C.sub.1-6 alkyl, OH, a C.sub.1-3 alkoxy, CF.sub.3, COCH.sub.3, and
COCF.sub.3 groups;
[0019] each of R.sub.2, R.sub.3, R.sub.4 and R.sub.5 is
independently selected from the group consisting of: H, hydroxyl,
halogen atom, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, alkynyl,
CF.sub.2R.sub.c, and OCF.sub.2R.sub.c groups, where R.sub.c is H, F
or alkyl; and
[0020] R.sub.6 is a group comprising a cyclic alkyl or aryl
moiety,
or a pharmaceutically acceptable form thereof.
[0021] In another aspect, the invention generally relates to a
pharmaceutical composition comprising a compound having the
structural formula of (I):
##STR00002##
wherein,
[0022] each of R.sub.a and R.sub.b is independently selected from
the group consisting of H, D and F;
[0023] L is a bivalent hydrocarbyl linker, optionally with one or
more carbon atoms replaced by a heteroatom selected from the group
consisting of O, S and N;
[0024] X is a halogen atom;
[0025] Y is N, O or S; provided that when Y is S or O, its bonding
to the adjacent carbons are single bonds;
[0026] Z is N--R.sub.1, O or S;
[0027] R.sub.1 is selected from the group consisting of: H, a
C.sub.1-6 alkyl, OH, a C.sub.1-3 alkoxy, CF.sub.3, COCH.sub.3, and
COCF.sub.3 groups;
[0028] each of R.sub.2, R.sub.3, R.sub.4 and R.sub.5 is
independently selected from the group consisting of: H, hydroxyl,
halogen atom, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, alkynyl,
CF.sub.2R.sub.c, and OCF.sub.2R.sub.c groups, where R.sub.c is H, F
or alkyl; and
[0029] R.sub.6 is a group comprising a cyclic alkyl or aryl
moiety,
or a pharmaceutically acceptable form thereof, effective to treat,
prevent, or reduce one or more diseases or disorders, in a mammal,
including a human, and a pharmaceutically acceptable excipient,
carrier, or diluent.
[0030] In yet another aspect, the invention generally relates to a
unit dosage form comprising a pharmaceutical composition disclosed
herein.
[0031] In yet another aspect, the invention generally relates to a
method for treating, reducing, or preventing a disease or disorder.
The method includes administering to a subject in need thereof a
pharmaceutical composition comprising a compound having the
structural formula of (I):
##STR00003##
wherein,
[0032] each of R.sub.a and R.sub.b is independently selected from
the group consisting of H, D and F;
[0033] L is a bivalent hydrocarbyl linker, optionally with one or
more carbon atoms replaced by a heteroatom selected from the group
consisting of O, S and N;
[0034] X is a halogen atom;
[0035] Y is N, O or S; provided that when Y is S or O, its bonding
to the adjacent carbons are single bonds;
[0036] Z is N--R.sub.1, O or S;
[0037] R.sub.1 is selected from the group consisting of: H, a
C.sub.1-6 alkyl, OH, a C.sub.1-3 alkoxy, CF.sub.3, COCH.sub.3, and
COCF.sub.3 groups;
[0038] each of R.sub.2, R.sub.3, R.sub.4 and R.sub.5 is
independently selected from the group consisting of: H, hydroxyl,
halogen atom, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, alkynyl,
CF.sub.2R.sub.c, and OCF.sub.2R.sub.c groups, where R.sub.c is H, F
or alkyl; and
[0039] R.sub.6 is a group comprising a cyclic alkyl or aryl
moiety,
or a pharmaceutically acceptable form thereof, and a
pharmaceutically acceptable excipient, carrier, or diluent,
effective to treat, prevent, or reduce one or more diseases or
disorders, in a mammal, including a human.
[0040] In yet another aspect, the invention generally relates to a
method for inhibiting or inactivating a protein arginine deiminase,
comprising administering to a subject in need thereof a
pharmaceutical composition comprising a compound having the
structural formula of (I):
##STR00004##
wherein,
[0041] each of R.sub.a and R.sub.b is independently selected from
the group consisting of H, D and F;
[0042] L is a bivalent hydrocarbyl linker, optionally with one or
more carbon atoms replaced by a heteroatom selected from the group
consisting of O, S and N;
[0043] X is a halogen atom;
[0044] Y is N, O or S; provided that when Y is S or O, its bonding
to the adjacent carbons are single bonds;
[0045] Z is N--R.sub.1, O or S;
[0046] R.sub.1 is selected from the group consisting of: H, a
C.sub.1-6 alkyl, OH, a C.sub.1-3 alkoxy, CF.sub.3, COCH.sub.3, and
COCF.sub.3 groups;
[0047] each of R.sub.2, R.sub.3, R.sub.4 and R.sub.5 is
independently selected from the group consisting of: H, hydroxyl,
halogen atom, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, alkynyl,
CF.sub.2R.sub.c and OCF.sub.2R.sub.c groups, where R.sub.c is H, F
or alkyl; and
[0048] R.sub.6 is a group comprising a cyclic alkyl or aryl
moiety,
or a pharmaceutically acceptable form thereof, and a
pharmaceutically acceptable excipient, carrier, or diluent,
effective to inhibit or inactivate a biological function of a
protein arginine deiminase, in a mammal, including a human.
[0049] In yet another aspect, the invention generally relates to a
molecular imaging probe having the structural formula:
A.sub.F-L.sub.F-W (II)
wherein
[0050] A.sub.F is a group comprising an optically detectable
moiety;
[0051] L.sub.F is a linking group; and
[0052] W is group comprising a benzimidazole moiety, or a
derivative or analog thereof, capable of inhibiting or inactivating
a biological function of a protein arginine deiminase.
[0053] In yet another aspect, the invention generally relates to a
method for identifying a protein arginine deiminase inhibitor or
inactivator. The method includes: performing a competitive assay
wherein a test compound competes with a molecular imaging probe
disclosed herein to bind to a protein arginine deiminase; and
measuring fluorescence to determine an amount of fluorescent
protein arginine deiminase present in the test assay.
BRIEF DESCRIPTION OF THE FIGURES
[0054] FIG. 1. (A) Concentration dependent labeling of recombinant
PAD2 with BB--F-Yne (5u). PAD2 was treated with increasing
concentrations of BB--F-Yne (5u) and then "Clicked" with
TAMRA-N.sub.3. (B) The limit of detection (LOD) of BB--F-Yne (5u)
for PAD2. Decreasing concentrations of PAD2 treated with BB--F-Yne
(5u) and "Clicked" with TAMRA-N.sub.3. The LOD was found to be 375
fmol. (C) Concentration dependent labeling of recombinant PAD2 with
BB--Cl-Yne (5v). PAD2 was treated with increasing concentrations of
BB--Cl-Yne (5) and then "Clicked" with TAMRA-N.sub.3. (D) The LOD
of BB--Cl-Yne (5v) for PAD2. Decreasing concentrations of PAD2 were
treated with BB--Cl-Yne (5v) and "Clicked" with TAMRA-N.sub.3.
[0055] FIG. 2. In vitro labeling of the four active PAD isozymes
with BB--F-Yne (5u). (A) Concentration dependent labeling of
recombinant PAD1 with BB--F-Yne (5u). PAD1 was treated with
increasing concentrations of BB--F-Yne (5u) and then "Clicked" with
TAMRA-N.sub.3. (B) Concentration dependent labeling of recombinant
PAD2 with BB--F-Yne (5u). PAD2 was treated with increasing
concentrations of BB--F-Yne (5u) and then "Clicked" with
TAMRA-N.sub.3. This data can also be seen in FIG. 1 and is repeated
here for clarity. (C) Concentration dependent labeling of
recombinant PAD3 with BB--F-Yne (5u). PAD3 was treated with
increasing concentrations of BB--F-Yne (5u) and then "Clicked" with
TAMRA-N.sub.3. (D) Concentration dependent labeling of recombinant
PAD4 with BB--F-Yne (5u). PAD4 was treated with increasing
concentrations of BB--F-Yne (5u) and then "Clicked" with
TAMRA-N.sub.3.
[0056] FIG. 3. Cellular labeling of PAD2 with BB--F-Yne (5u) in
ionophore stimulated HEK293T/PAD2 cells. (A) HEK293T/PAD2 cells
were treated with increasing concentrations of BB--F-Yne (5u) for 1
h. The cells were then harvested and probe labeled proteins were
tagged with TAMRA-N.sub.3 to facilitate visualization after
SDS-PAGE. (B&C) HEK293T/PAD2 cells were treated with increasing
concentrations of BB--F-Yne (5u) for 1 h. The cells were then
harvested and probe labeled proteins were tagged with
Biotin-TEV-N.sub.3. Biotin tagged proteins were then isolated on
streptavidin agarose and the eluted proteins were probed for PAD2
(panel B) or biotinylated proteins using streptavidin-HRP (panel
C).
[0057] FIG. 4. Cellular labeling of PAD2 with BB--Cl-Yne (5v) in
ionophore stimulated HEK293T/PAD2 cells. (A) HEK293T/PAD2 cells
were treated with increasing concentrations of BB--Cl-Yne (5) for 1
h. The cells were then harvested and probe labeled proteins were
tagged with TAMIRA-N.sub.3 to facilitate visualization after
SDS-PAGE. (B&C) HEK293T/PAD2 cells were treated with increasing
concentrations of BB--Cl-Yne (5v) for 1 h. The cells were then
harvested and probe labeled proteins were tagged with
Biotin-TEV-N.sub.3. Biotin tagged proteins were then isolated on
streptavidin agarose and the eluted proteins were probed for PAD2
(panel B) or biotinylated proteins using streptavidin-HRP (panel
C).
[0058] FIG. 5. Cellular labeling of PAD4 with BB--F-Yne (5u). (A)
HEK293T/PAD4 cells were treated with increasing concentrations of
BB--F-Yne (5u) for 1 h. The cells were then harvested and probe
labeled proteins were tagged with TAMIRA-N.sub.3 to facilitate
visualization after SDS-PAGE. (B) HEK293T/PAD4 cells were treated
with increasing concentrations of BB--F-Yne (5u) for 1 h. The cells
were then harvested and probe labeled proteins were tagged with
Biotin-TEV-N.sub.3. Biotin tagged proteins were then isolated on
streptavidin agarose and the eluted proteins were probed for
PAD2.
DEFINITIONS
[0059] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. General
principles of organic chemistry, as well as specific functional
moieties and reactivity, are described in "Organic Chemistry",
Thomas Sorrell, University Science Books, Sausalito: 2006.
[0060] Certain compounds of the present invention may exist in
particular geometric or stereoisomeric forms. The present invention
contemplates all such compounds, including cis- and trans-isomers,
R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the
racemic mixtures thereof, and other mixtures thereof, as falling
within the scope of the invention. Additional asymmetric carbon
atoms may be present in a substituent such as an alkyl group. All
such isomers, as well as mixtures thereof, are intended to be
included in this invention.
[0061] Isomeric mixtures containing any of a variety of isomer
ratios may be utilized in accordance with the present invention.
For example, where only two isomers are combined, mixtures
containing 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3,
98:2, 99:1, or 100:0 isomer ratios are contemplated by the present
invention. Those of ordinary skill in the art will readily
appreciate that analogous ratios are contemplated for more complex
isomer mixtures.
[0062] If, for instance, a particular enantiomer of a compound of
the present invention is desired, it may be prepared by asymmetric
synthesis, or by derivation with a chiral auxiliary, where the
resulting diastereomeric mixture is separated and the auxiliary
group cleaved to provide the pure desired enantiomers.
Alternatively, where the molecule contains a basic functional
group, such as amino, or an acidic functional group, such as
carboxyl, diastereomeric salts are formed with an appropriate
optically-active acid or base, followed by resolution of the
diastereomers thus formed by fractional crystallization or
chromatographic methods well known in the art, and subsequent
recovery of the pure enantiomers.
[0063] As used herein, "administration" of a disclosed compound
encompasses the delivery to a subject of a compound as described
herein, or a prodrug or other pharmaceutically acceptable
derivative thereof, using any suitable formulation or route of
administration, as discussed herein.
[0064] As used herein, the terms "effective amount" or
"therapeutically effective amount" refer to that amount of a
compound or pharmaceutical composition described herein that is
sufficient to effect the intended application including, but not
limited to, disease treatment, as illustrated below.
[0065] In some embodiments, the amount is that effective for stop
the progression or effect reduction of an inflammatory disease or
disorder. In some embodiments, the amount is that effective for
stop the progression or effect reduction of an immune system
disorders. In some embodiments, the amount is that effective to
stop the progression or effect reduction of an autoimmune disease
or disorder. In some embodiments, the amount is that effective for
stop the progression or effect reduction of a cardiovascular
disease or disorder. In some embodiments, the amount is that
effective for detectable killing or inhibition of the growth or
spread of cancer cells; the size or number of tumors; or other
measure of the level, stage, progression or severity of the
cancer.
[0066] The therapeutically effective amount can vary depending upon
the intended application, or the subject and disease condition
being treated, e.g., the desired biological endpoint, the
pharmacokinetics of the compound, the disease being treated, the
mode of administration, and the weight and age of the patient,
which can readily be determined by one of ordinary skill in the
art. The term also applies to a dose that will induce a particular
response in target cells, e.g., reduction of cell migration. The
specific dose will vary depending on, for example, the particular
compounds chosen, the species of subject and their age/existing
health conditions or risk for health conditions, the dosing regimen
to be followed, the severity of the disease, whether it is
administered in combination with other agents, timing of
administration, the tissue to which it is administered, and the
physical delivery system in which it is carried.
[0067] As used herein, the terms "treatment" or "treating" a
disease or disorder refers to a method of reducing, delaying or
ameliorating such a condition before or after it has occurred.
Treatment may be directed at one or more effects or symptoms of a
disease and/or the underlying pathology. Treatment is aimed to
obtain beneficial or desired results including, but not limited to,
therapeutic benefit and/or a prophylactic benefit. By therapeutic
benefit is meant eradication or amelioration of the underlying
disorder being treated. Also, a therapeutic benefit is achieved
with the eradication or amelioration of one or more of the
physiological symptoms associated with the underlying disorder such
that an improvement is observed in the patient, notwithstanding
that the patient can still be afflicted with the underlying
disorder. For prophylactic benefit, the pharmaceutical compounds
and/or compositions can be administered to a patient at risk of
developing a particular disease, or to a patient reporting one or
more of the physiological symptoms of a disease, even though a
diagnosis of this disease may not have been made. The treatment can
be any reduction and can be, but is not limited to, the complete
ablation of the disease or the symptoms of the disease. As compared
with an equivalent untreated control, such reduction or degree of
prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%,
or 100% as measured by any standard technique.
[0068] As used herein, the term "therapeutic effect" refers to a
therapeutic benefit and/or a prophylactic benefit as described
herein. A prophylactic effect includes delaying or eliminating the
appearance of a disease or condition, delaying or eliminating the
onset of symptoms of a disease or condition, slowing, halting, or
reversing the progression of a disease or condition, or any
combination thereof.
[0069] As used herein, a "pharmaceutically acceptable form" of a
disclosed compound includes, but is not limited to,
pharmaceutically acceptable salts, esters, hydrates, solvates,
isomers, prodrugs, and isotopically labeled derivatives of
disclosed compounds. In one embodiment, a "pharmaceutically
acceptable form" includes, but is not limited to, pharmaceutically
acceptable salts, esters, isomers, prodrugs and isotopically
labeled derivatives of disclosed compounds. In some embodiments, a
"pharmaceutically acceptable form" includes, but is not limited to,
pharmaceutically acceptable salts, esters, stereoisomers, prodrugs
and isotopically labeled derivatives of disclosed compounds.
[0070] In certain embodiments, the pharmaceutically acceptable form
is a pharmaceutically acceptable salt. As used herein, the term
"pharmaceutically acceptable salt" refers to those salts which are,
within the scope of sound medical judgment, suitable for use in
contact with the tissues of subjects without undue toxicity,
irritation, allergic response and the like, and are commensurate
with a reasonable benefit/risk ratio. Pharmaceutically acceptable
salts are well known in the art. For example, Berge et al.
describes pharmaceutically acceptable salts in detail in J.
Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptable
salts of the compounds provided herein include those derived from
suitable inorganic and organic acids and bases. Examples of
pharmaceutically acceptable, nontoxic acid addition salts are salts
of an amino group formed with inorganic acids such as hydrochloric
acid, hydrobromic acid, phosphoric acid, sulfuric acid and
perchioric acid or with organic acids such as acetic acid, oxalic
acid, maleic acid, tartaric acid, citric acid, succinic acid or
malonic acid or by using other methods used in the art such as ion
exchange. Other pharmaceutically acceptable salts include adipate,
alginate, ascorbate, aspartate, benzenesulfonate, besylate,
benzoate, bisulfate, borate, butyrate, camphorate,
camphorsulfonate, citrate, cyclopentanepropionate, digluconate,
dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate,
glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate,
hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate,
laurate, lauryl sulfate, malate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
oleate, oxalate, palmitate, pamoate, pectinate, persulfate,
3-phenylpropionate, phosphate, picrate, pivalate, propionate,
stearate, succinate, sulfate, tartrate, thiocyanate,
p-toluenesulfonate, undecanoate, valerate salts, and the like. In
some embodiments, organic acids from which salts can be derived
include, for example, acetic acid, propionic acid, glycolic acid,
pyruvic acid, oxalic acid, lactic acid, trifluoracetic acid, maleic
acid, malonic acid, succinic acid, fumaric acid, tartaric acid,
citric acid, benzoic acid, cinnamic acid, mandelic acid,
methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,
salicylic acid, and the like.
[0071] The salts can be prepared in situ during the isolation and
purification of the disclosed compounds, or separately, such as by
reacting the free base or free acid of a parent compound with a
suitable base or acid, respectively. Pharmaceutically acceptable
salts derived from appropriate bases include alkali metal, alkaline
earth metal, ammonium and N+(C.sub.1-4alkyl).sup.4 salts.
Representative alkali or alkaline earth metal salts include sodium,
lithium, potassium, calcium, magnesium, iron, zinc, copper,
manganese, aluminum, and the like. Further pharmaceutically
acceptable salts include, when appropriate, nontoxic ammonium,
quaternary ammonium, and amine cations formed using counterions
such as halide, hydroxide, carboxylate, sulfate, phosphate,
nitrate, lower alkyl sulfonate and aryl sulfonate. Organic bases
from which salts can be derived include, for example, primary,
secondary, and tertiary amines, substituted amines, including
naturally occurring substituted amines, cyclic amines, basic ion
exchange resins, and the like, such as isopropylamine,
trimethylamine, diethylamine, triethylamine, tripropylamine, and
ethanolamine. In some embodiments, the pharmaceutically acceptable
base addition salt can be chosen from ammonium, potassium, sodium,
calcium, and magnesium salts.
[0072] In certain embodiments, the pharmaceutically acceptable form
is a pharmaceutically acceptable ester. As used herein, the term
"pharmaceutically acceptable ester" refers to esters that hydrolyze
in vivo and include those that break down readily in the human body
to leave the parent compound or a salt thereof. Such esters can act
as a prodrug as defined herein. Pharmaceutically acceptable esters
include, but are not limited to, alkyl, alkenyl, alkynyl, aryl,
aralkyl, and cycloalkyl esters of acidic groups, including, but not
limited to, carboxylic acids, phosphoric acids, phosphinic acids,
sulfinic acids, sulfonic acids and boronic acids. Examples of
esters include formates, acetates, propionates, butyrates,
acrylates and ethylsuccinates. The esters can be formed with a
hydroxy or carboxylic acid group of the parent compound.
[0073] As used herein, the term "pharmaceutically acceptable enol
ethers" include, but are not limited to, derivatives of formula
--C.dbd.C(OR) where R can be selected from alkyl, alkenyl, alkynyl,
aryl, aralkyl and cycloalkyl. Pharmaceutically acceptable enol
esters include, but are not limited to, derivatives of formula
--C.dbd.C(OC(O)R) where R can be selected from hydrogen, alkyl,
alkenyl, alkynyl, aryl, aralkyl and cycloalkyl.
[0074] In certain embodiments, the pharmaceutically acceptable form
is a "solvate" (e.g., a hydrate). As used herein, the term
"solvate" refers to compounds that further include a stoichiometric
or non-stoichiometric amount of solvent bound by non-covalent
intermolecular forces. The solvate can be of a disclosed compound
or a pharmaceutically acceptable salt thereof. Where the solvent is
water, the solvate is a "hydrate". Pharmaceutically acceptable
solvates and hydrates are complexes that, for example, can include
1 to about 100, or 1 to about 10, or 1 to about 2, about 3 or about
4, solvent or water molecules. It will be understood that the term
"compound" as used herein encompasses the compound and solvates of
the compound, as well as mixtures thereof.
[0075] In certain embodiments, the pharmaceutically acceptable form
is a prodrug. As used herein, the term "prodrug" (or "pro-drug")
refers to compounds that are transformed in vivo to yield a
disclosed compound or a pharmaceutically acceptable form of the
compound. A prodrug can be inactive when administered to a subject,
but is converted in vivo to an active compound, for example, by
hydrolysis (e.g., hydrolysis in blood). In certain cases, a prodrug
has improved physical and/or delivery properties over the parent
compound. Prodrugs can increase the bioavailability of the compound
when administered to a subject (e.g., by permitting enhanced
absorption into the blood following oral administration) or which
enhance delivery to a biological compartment of interest (e.g., the
brain or lymphatic system) relative to the parent compound.
Exemplary prodrugs include derivatives of a disclosed compound with
enhanced aqueous solubility or active transport through the gut
membrane, relative to the parent compound.
[0076] The prodrug compound often offers advantages of solubility,
tissue compatibility or delayed release in a mammalian organism
(see, e.g., Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24
(Elsevier, Amsterdam). A discussion of prodrugs is provided in
Higuchi, T., et al., "Prodrugs as Novel Delivery Systems," A.C.S.
Symposium Series, Vol. 14, and in Bioreversible Carriers in Drug
Design, ed. Edward B. Roche, American Pharmaceutical Association
and Pergamon Press, 1987, both of which are incorporated in full by
reference herein. Exemplary advantages of a prodrug can include,
but are not limited to, its physical properties, such as enhanced
water solubility for parenteral administration at physiological pH
compared to the parent compound, or it can enhance absorption from
the digestive tract, or it can enhance drug stability for long-term
storage.
[0077] As used herein, the term "pharmaceutically acceptable"
excipient, carrier, or diluent refers to a pharmaceutically
acceptable material, composition or vehicle, such as a liquid or
solid filler, diluent, excipient, solvent or encapsulating
material, involved in carrying or transporting the subject
pharmaceutical agent from one organ, or portion of the body, to
another organ, or portion of the body. Each carrier must be
"acceptable" in the sense of being compatible with the other
ingredients of the formulation and not injurious to the patient.
Some examples of materials which can serve as
pharmaceutically-acceptable carriers include: sugars, such as
lactose, glucose and sucrose; starches, such as corn starch and
potato starch; cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate;
powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa
butter and suppository waxes; oils, such as peanut oil, cottonseed
oil, safflower oil, sesame oil, olive oil, corn oil and soybean
oil; glycols, such as propylene glycol; polyols, such as glycerin,
sorbitol, mannitol and polyethylene glycol; esters, such as ethyl
oleate and ethyl laurate; agar; buffering agents, such as magnesium
hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;
isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer
solutions; and other non-toxic compatible substances employed in
pharmaceutical formulations. Wetting agents, emulsifiers and
lubricants, such as sodium lauryl sulfate, magnesium stearate, and
polyethylene oxide-polypropylene oxide copolymer as well as
coloring agents, release agents, coating agents, sweetening,
flavoring and perfuming agents, preservatives and antioxidants can
also be present in the compositions.
[0078] As used herein, the term "subject" refers to any animal
(e.g., a mammal), including, but not limited to humans, non-human
primates, rodents, and the like, which is to be the recipient of a
particular treatment. Typically, the terms "subject" and "patient"
are used interchangeably herein in reference to a human
subject.
[0079] Compounds of the present invention are, subsequent to their
preparation, preferably isolated and purified to obtain a
composition containing an amount by weight equal to or greater than
95% ("substantially pure"), which is then used or formulated as
described herein. In certain embodiments, the compounds of the
present invention are more than 99% pure.
[0080] Solvates and polymorphs of the compounds of the invention
are also contemplated herein. Solvates of the compounds of the
present invention include, for example, hydrates.
[0081] Definitions of specific functional groups and chemical terms
are described in more detail below. When a range of values is
listed, it is intended to encompass each value and sub-range within
the range. For example "C.sub.1-6 alkyl" is intended to encompass,
C.sub.1, C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6, C.sub.1-6,
C.sub.1-5, C.sub.1-4, C.sub.1-3, C.sub.1-2, C.sub.2-6, C.sub.2-5,
C.sub.2-4, C.sub.2-3, C.sub.3-6, C.sub.3-5, C.sub.3-4, C.sub.4-6,
C.sub.4-5, and C.sub.5-6 alkyl.
[0082] As used herein, the term "alkyl" refers to a straight or
branched hydrocarbon chain radical consisting solely of carbon and
hydrogen atoms, containing no unsaturation, having from one to ten
carbon atoms (e.g., C.sub.1-10 alkyl). Whenever it appears herein,
a numerical range such as "1 to 10" refers to each integer in the
given range; e.g., "1 to 10 carbon atoms" means that the alkyl
group can consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms,
etc., up to and including 10 carbon atoms, although the present
definition also covers the occurrence of the term "alkyl" where no
numerical range is designated. In some embodiments, "alkyl" can be
a C.sub.1-6 alkyl group. In some embodiments, alkyl groups have 1
to 10, 1 to 8, 1 to 6, or 1 to 3 carbon atoms. Representative
saturated straight chain alkyls include, but are not limited to,
-methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and -n-hexyl;
while saturated branched alkyls include, but are not limited to,
-isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl,
2-methylbutyl, 3-methylbutyl, 2-methylpentyl, 3-methylpentyl,
4-methylpentyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl,
5-methylhexyl, 2,3-dimethylbutyl, and the like. The alkyl is
attached to the parent molecule by a single bond. Unless stated
otherwise in the specification, an alkyl group is optionally
substituted by one or more of substituents which independently
include: acyl, alkyl, alkenyl, alkynyl, alkoxy, alkylaryl,
cycloalkyl, aralkyl, aryl, aryloxy, amino, amido, amidino, imino,
azide, carbonate, carbamate, carbonyl, heteroalkyl, heteroaryl,
heteroarylalkyl, heterocycloalkyl, hydroxy, cyano, halo,
haloalkoxy, haloalkyl, ester, ether, mercapto, thio, alkylthio,
arylthio, thiocarbonyl, nitro, oxo, phosphate, phosphonate,
phosphinate, silyl, sulfinyl, sulfonyl, sulfonamidyl, sulfoxyl,
sulfonate, urea, --Si(R.sup.a).sub.3, --OR.sup.a, --SR.sup.a,
--OC(O)--R.sup.a, --N(R.sup.a).sub.2, --C(O)R.sub.a,
--C(O)OR.sup.a, --OC(O)N(R.sup.a).sub.2, --C(O)N(R.sup.a).sub.2,
--N(R.sup.a)C(O)OR.sup.a, --N(R.sup.a)C(O)R.sup.a,
--N(R.sup.a)C(O)N(R.sup.a).sub.2,
--N(R.sup.a)C(NR.sup.a)N(R.sup.a).sub.2,
--N(R.sup.a)S(O).sub.tN(R.sup.a).sub.2 (where t is 1 or 2),
--P(.dbd.O)(R.sup.a)(R.sup.a), or --O--P(.dbd.O)(OR.sup.a).sub.2
where each R.sup.a is independently hydrogen, alkyl, haloalkyl,
carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,
heterocycloalkylalkyl, heteroaryl or heteroarylalkyl, and each of
these moieties can be optionally substituted as defined herein. In
a non-limiting embodiment, a substituted alkyl can be selected from
fluoromethyl, difluoromethyl, trifluoromethyl, 2-fluoroethyl,
3-fluoropropyl, hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl,
benzyl, and phenethyl.
[0083] As used herein, the term "alkoxy" refers to the group
--O-alkyl, including from 1 to 10 carbon atoms (C.sub.1-10) of a
straight, branched, saturated cyclic configuration and combinations
thereof, attached to the parent molecular structure through an
oxygen. Examples include methoxy, ethoxy, propoxy, isopropoxy,
butoxy, t-butoxy, pentoxy, cyclopropyloxy, cyclohexyloxy and the
like. "Lower alkoxy" refers to alkoxy groups containing one to six
carbons. In some embodiments, C.sub.1-3 alkoxy is an alkoxy group
that encompasses both straight and branched chain alkyls of from 1
to 3 carbon atoms. Unless stated otherwise in the specification, an
alkoxy group can be optionally substituted by one or more
substituents which independently include: acyl, alkyl, alkenyl,
alkynyl, alkoxy, alkylaryl, cycloalkyl, aralkyl, aryl, aryloxy,
amino, amido, amidino, imino, azide, carbonate, carbamate,
carbonyl, heteroalkyl, heteroaryl, heteroarylalkyl,
heterocycloalkyl, hydroxy, cyano, halo, haloalkoxy, haloalkyl,
ester, ether, mercapto, thio, alkylthio, arylthio, thiocarbonyl,
nitro, oxo, phosphate, phosphonate, phosphinate, silyl, sulfinyl,
sulfonyl, sulfonamidyl, sulfoxyl, sulfonate, urea,
--Si(R.sup.a).sub.3, --OR.sup.a, --SR.sup.a, --OC(O)--R.sup.a,
--N(R.sup.a).sub.2, --C(O)R.sup.a, --C(O)OR.sup.a,
--OC(O)N(R.sup.a).sub.2, --C(O)N(R.sup.a).sub.2,
--N(R.sup.a)C(O)OR.sup.a, --N(R.sup.a)C(O)R.sup.a,
--N(R.sup.a)C(O)N(R.sup.a).sub.2,
--N(R.sup.a)C(NR.sup.a)N(R.sup.a).sub.2,
--N(R.sup.a)S(O).sub.tN(R.sup.a).sub.2 (where t is 1 or 2),
--P(.dbd.O)(R.sup.a)(R.sup.a), or --O--P(.dbd.O)(OR.sup.a).sub.2
where each R.sup.a is independently hydrogen, alkyl, haloalkyl,
carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,
heterocycloalkylalkyl, heteroaryl or heteroarylalkyl, and each of
these moieties can be optionally substituted as defined herein.
[0084] As used herein, the terms "aromatic" or "aryl" refer to a
radical with 6 to 14 ring atoms (e.g., C.sub.6-14 aromatic or
C.sub.6-14 aryl) which has at least one ring having a conjugated pi
electron system which is carbocyclic (e.g., phenyl, fluorenyl, and
naphthyl). In some embodiments, the aryl is a C.sub.6-10 aryl
group. For example, bivalent radicals formed from substituted
benzene derivatives and having the free valences at ring atoms are
named as substituted phenylene radicals. In other embodiments,
bivalent radicals derived from univalent polycyclic hydrocarbon
radicals whose names end in"-yl" by removal of one hydrogen atom
from the carbon atom with the free valence are named by adding
"-idene" to the name of the corresponding univalent radical, e.g.,
a naphthyl group with two points of attachment is termed
naphthylidene. Whenever it appears herein, a numerical range such
as "6 to 14 aryl" refers to each integer in the given range; e.g.,
"6 to 14 ring atoms" means that the aryl group can consist of 6
ring atoms, 7 ring atoms, etc., up to and including 14 ring atoms.
The term includes monocyclic or fused-ring polycyclic (i.e., rings
which share adjacent pairs of ring atoms) groups. Polycyclic aryl
groups include bicycles, tricycles, tetracycles, and the like. In a
multi-ring group, only one ring is required to be aromatic, so
groups such as indanyl are encompassed by the aryl definition.
Non-limiting examples of aryl groups include phenyl, phenalenyl,
naphthalenyl, tetrahydronaphthyl, phenanthrenyl, anthracenyl,
fluorenyl, indolyl, indanyl, and the like. Unless stated otherwise
in the specification, an aryl moiety can be optionally substituted
by one or more substituents which independently include: acyl,
alkyl, alkenyl, alkynyl, alkoxy, alkylaryl, cycloalkyl, aralkyl,
aryl, aryloxy, amino, amido, amidino, imino, azide, carbonate,
carbamate, carbonyl, heteroalkyl, heteroaryl, heteroarylalkyl,
heterocycloalkyl, hydroxy, cyano, halo, haloalkoxy, haloalkyl,
ester, ether, mercapto, thio, alkylthio, arylthio, thiocarbonyl,
nitro, oxo, phosphate, phosphonate, phosphinate, silyl, sulfinyl,
sulfonyl, sulfonamidyl, sulfoxyl, sulfonate, urea,
--Si(R.sup.a).sub.3--OR.sup.a, --SR.sup.a, --OC(O)--R.sup.a,
--N(R.sup.a).sub.2, --C(O)R.sub.a, --C(O)OR.sup.a,
--OC(O)N(R.sup.a).sub.2, --C(O)N(R.sup.a).sub.2,
--N(R.sup.a)C(O)OR.sup.a, --N(R.sup.a)C(O)R.sup.a,
--N(R.sup.a)C(O)N(R.sup.a).sub.2,
--N(R.sup.a)C(NR.sup.a)N(R.sup.a).sub.2,
--N(R.sup.a)S(O).sub.tN(R.sup.a).sub.2 (where t is 1 or 2),
--P(.dbd.O)(R.sup.a)(R.sup.a), or --O--P(.dbd.O)(OR.sup.a).sub.2
where each R.sup.a is independently hydrogen, alkyl, haloalkyl,
carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,
heterocycloalkylalkyl, heteroaryl or heteroarylalkyl, and each of
these moieties can be optionally substituted as defined herein.
[0085] As used herein, the terms "cycloalkyl" and "carbocyclyl"
each refers to a monocyclic or polycyclic radical that contains
only carbon and hydrogen, and can be saturated or partially
unsaturated. Partially unsaturated cycloalkyl groups can be termed
"cycloalkenyl" if the carbocycle contains at least one double bond,
or "cycloalkynyl" if the carbocycle contains at least one triple
bond. Cycloalkyl groups include groups having from 3 to 13 ring
atoms (i.e., C.sub.3-13 cycloalkyl). Whenever it appears herein, a
numerical range such as "3 to 10" refers to each integer in the
given range; e.g., "3 to 13 carbon atoms" means that the cycloalkyl
group can consist of 3 carbon atoms, 4 carbon atoms, 5 carbon
atoms, etc., up to and including 13 carbon atoms. The term
"cycloalkyl" also includes bridged and spiro-fused cyclic
structures containing no heteroatoms. The term also includes
monocyclic or fused-ring polycyclic (i.e., rings which share
adjacent pairs of ring atoms) groups. Polycyclic aryl groups
include bicycles, tricycles, tetracycles, and the like. In some
embodiments, "cycloalkyl" can be a C.sub.3-8 cycloalkyl radical. In
some embodiments, "cycloalkyl" can be a C.sub.3-5 cycloalkyl
radical. Illustrative examples of cycloalkyl groups include, but
are not limited to the following moieties: C.sub.3-6 carbocyclyl
groups include, without limitation, cyclopropyl (C.sub.3),
cyclobutyl (C.sub.4), cyclopentyl (C.sub.5), cyclopentenyl
(C.sub.5), cyclohexyl (C.sub.6), cyclohexenyl (C.sub.6),
cyclohexadienyl (C.sub.6) and the like. Examples of C.sub.3-7
carbocyclyl groups include norbornyl (C.sub.7). Examples of
C.sub.3-8 carbocyclyl groups include the aforementioned C.sub.3-7
carbocyclyl groups as well as cycloheptyl (C.sub.7),
cycloheptadienyl (C.sub.7), cycloheptatrienyl (C.sub.7), cyclooctyl
(C.sub.8), bicyclo[2.2.1]heptanyl, bicyclo[2.2.2]octanyl, and the
like. Examples of C.sub.3-13 carbocyclyl groups include the
aforementioned C.sub.3-8 carbocyclyl groups as well as octahydro-1H
indenyl, decahydronaphthalenyl, spiro[4.5]decanyl and the like.
Unless stated otherwise in the specification, a cycloalkyl group
can be optionally substituted by one or more substituents which
independently include: acyl, alkyl, alkenyl, alkynyl, alkoxy,
alkylaryl, cycloalkyl, aralkyl, aryl, aryloxy, amino, amido,
amidino, imino, azide, carbonate, carbamate, carbonyl, heteroalkyl,
heteroaryl, heteroarylalkyl, heterocycloalkyl, hydroxy, cyano,
halo, haloalkoxy, haloalkyl, ester, ether, mercapto, thio,
alkylthio, arylthio, thiocarbonyl, nitro, oxo, phosphate,
phosphonate, phosphinate, silyl, sulfinyl, sulfonyl, sulfonamidyl,
sulfoxyl, sulfonate, urea, --Si(R.sup.a).sub.3, --OR.sup.a,
--SR.sup.a, --OC(O)--R.sup.a, --N(R.sup.a).sub.2, --C(O)R.sup.a,
--C(O)OR.sup.a, --OC(O)N(R.sup.a).sub.2, --C(O)N(R.sup.a).sub.2,
--N(R.sup.a)C(O)OR.sup.a, --N(R.sup.a)C(O)R.sup.a,
--N(R.sup.a)C(O)N(R.sup.a).sub.2,
--N(R.sup.a)C(NR.sup.a)N(R.sup.a).sub.2,
--N(R.sup.a)S(O).sub.tN(R.sup.a).sub.2 (where t is 1 or 2),
--P(.dbd.O)(R.sup.a)(R.sup.a), or --O--P(.dbd.O)(OR.sup.a).sub.2
where each R.sup.a is independently hydrogen, alkyl, haloalkyl,
carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,
heterocycloalkylalkyl, heteroaryl or heteroarylalkyl, and each of
these moieties can be optionally substituted as defined herein. The
terms "cycloalkenyl" and "cycloalkynyl" mirror the above
description of "cycloalkyl" wherein the prefix "alk" is replaced
with "alken" or "alkyn" respectively, and the parent "alkenyl" or
"alkynyl" terms are as described herein. For example, a
cycloalkenyl group can have 3 to 13 ring atoms, such as 5 to 8 ring
atoms. In some embodiments, a cycloalkynyl group can have 5 to 13
ring atoms.
[0086] As used herein, the term "halide", "halo", or,
alternatively, "halogen" means fluoro, chioro, bromo or iodo. The
terms "haloalkyl," "haloalkenyl," "haloalkynyl" and "haloalkoxy"
include alkyl, alkenyl, alkynyl and alkoxy structures that are
substituted with one or more halo groups or with combinations
thereof. For example, the terms "fluoroalkyl" and "fluoroalkoxy"
include haloalkyl and haloalkoxy groups, respectively, in which the
halo is fluorine, such as, but not limited to, trifluoromethyl,
difluoromethyl, 2,2,2-trifluoroethyl, 1-fluoromethyl-2-fluoroethyl,
and the like. Each of the alkyl, alkenyl, alkynyl and alkoxy groups
are as defined herein and can be optionally further substituted as
defined herein.
[0087] As used herein, the term "heteroalkyl" refers to an alkyl
radical, which have one or more skeletal chain atoms selected from
an atom other than carbon, e.g., oxygen, nitrogen, sulfur,
phosphorus or combinations thereof. A numerical range can be given,
e.g., C.sub.1-4 heteroalkyl which refers to the chain length in
total, which in this example is 4 atoms long. For example, a
--CH.sub.2OCH.sub.2CH.sub.3 radical is referred to as a "C.sub.4"
heteroalkyl, which includes the heteroatom center in the atom chain
length description. Connection to the parent molecular structure
can be through either a heteroatom or a carbon in the heteroalkyl
chain. For example, an N-containing heteroalkyl moiety refers to a
group in which at least one of the skeletal atoms is a nitrogen
atom. One or more heteroatom(s) in the heteroalkyl radical can be
optionally oxidized. One or more nitrogen atoms, if present, can
also be optionally quaternized. For example, heteroalkyl also
includes skeletal chains substituted with one or more nitrogen
oxide (--O--) substituents. Exemplary heteroalkyl groups include,
without limitation, ethers such as methoxyethanyl
(--CH.sub.2CH.sub.2OCH.sub.3), ethoxymethanyl
(--CH.sub.2OCH.sub.2CH.sub.3), (methoxymethoxy)ethanyl
(--CH.sub.2CH.sub.2OCH.sub.2OCH.sub.3), (methoxymethoxy) methanyl
(--CH.sub.2OCH.sub.2OCH.sub.3) and (methoxyethoxy)methanyl
(--CH.sub.2OCH.sub.2CH.sub.2OCH.sub.3) and the like; amines such as
(--CH.sub.2CH.sub.2NHCH.sub.3, --CH.sub.2CH.sub.2N(CH.sub.3).sub.2,
--CH.sub.2NHCH.sub.2CH.sub.3,
--CH.sub.2N(CH.sub.2CH.sub.3)(CH.sub.3)) and the like.
[0088] As used herein, the term "heteroaryl" or, alternatively,
"heteroaromatic" refers to a refers to a radical of a 5-18 membered
monocyclic or polycyclic (e.g., bicyclic, tricyclic, tetracyclic
and the like) aromatic ring system (e.g., having 6, 10 or 14 .pi.
electrons shared in a cyclic array) having ring carbon atoms and
1-6 ring heteroatoms provided in the aromatic ring system, wherein
each heteroatom is independently selected from nitrogen, oxygen,
phosphorous and sulfur ("5-18 membered heteroaryl"). Heteroaryl
polycyclic ring systems can include one or more heteroatoms in one
or both rings. Whenever it appears herein, a numerical range such
as "5 to 18" refers to each integer in the given range; e.g., "5 to
18 ring atoms" means that the heteroaryl group can consist of 5
ring atoms, 6 ring atoms, etc., up to and including 18 ring atoms.
In some instances, a heteroaryl can have 5 to 14 ring atoms. In
some embodiments, the heteroaryl has, for example, bivalent
radicals derived from univalent heteroaryl radicals whose names end
in "-yl" by removal of one hydrogen atom from the atom with the
free valence are named by adding "-ene" to the name of the
corresponding univalent radical, e.g., a pyridyl group with two
points of attachment is a pyridylene.
[0089] For example, an N-containing "heteroaromatic" or
"heteroaryl" moiety refers to an aromatic group in which at least
one of the skeletal atoms of the ring is a nitrogen atom. One or
more heteroatom(s) in the heteroaryl radical can be optionally
oxidized. One or more nitrogen atoms, if present, can also be
optionally quaternized. Heteroaryl also includes ring systems
substituted with one or more nitrogen oxide (--O--) substituents,
such as pyridinyl N-oxides. The heteroaryl is attached to the
parent molecular structure through any atom of the ring(s).
[0090] "Heteroaryl" also includes ring systems wherein the
heteroaryl ring, as defined above, is fused with one or more aryl
groups wherein the point of attachment to the parent molecular
structure is either on the aryl or on the heteroaryl ring, or
wherein the heteroaryl ring, as defined above, is fused with one or
more cycloalkyl or heterocycyl groups wherein the point of
attachment to the parent molecular structure is on the heteroaryl
ring. For polycyclic heteroaryl groups wherein one ring does not
contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl and the
like), the point of attachment to the parent molecular structure
can be on either ring, i.e., either the ring bearing a heteroatom
(e.g., 2-indolyl) or the ring that does not contain a heteroatom
(e.g., 5-indolyl). In some embodiments, a heteroaryl group is a
5-10 membered aromatic ring system having ring carbon atoms and 1-4
ring heteroatoms provided in the aromatic ring system, wherein each
heteroatom is independently selected from nitrogen, oxygen,
phosphorous, and sulfur ("5-10 membered heteroaryl"). In some
embodiments, a heteroaryl group is a 5-8 membered aromatic ring
system having ring carbon atoms and 1-4 ring heteroatoms provided
in the aromatic ring system, wherein each heteroatom is
independently selected from nitrogen, oxygen, phosphorous, and
sulfur ("5-8 membered heteroaryl"). In some embodiments, a
heteroaryl group is a 5-6 membered aromatic ring system having ring
carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring
system, wherein each heteroatom is independently selected from
nitrogen, oxygen, phosphorous, and sulfur ("5-6 membered
heteroaryl"). In some embodiments, the 5-6 membered heteroaryl has
1-3 ring heteroatoms selected from nitrogen, oxygen, phosphorous,
and sulfur. In some embodiments, the 5-6 membered heteroaryl has
1-2 ring heteroatoms selected from nitrogen, oxygen, phosphorous,
and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1
ring heteroatom selected from nitrogen, oxygen, phosphorous, and
sulfur.
[0091] Examples of heteroaryls include, but are not limited to,
azepinyl, acridinyl, benzimidazolyl, benzindolyl,
1,3-benzodioxolyl, benzofuranyl, benzooxazolyl, benzo[d]thiazolyl,
benzothiadiazolyl, benzo[b][1,4]dioxepinyl, benzo[b][1,4] oxazinyl,
1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl,
benzodioxolyl, benzodioxinyl, benzoxazolyl, benzopyranyl,
benzopyranonyl, benzofuranyl, benzopyranonyl, benzofurazanyl,
benzothiazolyl, benzothienyl (benzothiophenyl),
benzothieno[3,2-d]pyrimidinyl, benzotriazolyl,
benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl,
cyclopenta[d]pyrimidinyl, 6,7-dihydro-5H-cyclopenta[4,5]thieno
[2,3-d]pyrimidinyl, 5,6-dihydrobenzo[h]quinazolinyl,
5,6-dihydrobenzo[h]cinnolinyl, 6,7-dihydro-5H
benzo[6,7]cyclohepta[1,2-c]pyridazinyl, dibenzofuranyl,
dibenzothiophenyl, furanyl, furazanyl, furanonyl, furo
[3,2-c]pyridinyl, 5,6,7,8,9,10-hexahydrocycloocta[d] pyrimidinyl,
5,6,7,8,9,10-hexahydrocycloocta[d]pyridazinyl,
5,6,7,8,9,10-hexahydrocycloocta[d]pyridinyl, isothiazolyl,
imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl,
isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl,
5,8-methano-5,6,7,8-tetrahydroquinazolinyl, naphthyridinyl,
1,6-naphthyridinonyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl,
oxiranyl, 5,6,6a,7,8,9,10,10a-octahydrobenzo[h]quinazolinyl,
1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl,
phthalazinyl, pteridinyl, purinyl, pyranyl, pyrrolyl, pyrazolyl,
pyrazolo[3,4-d]pyrimidinyl, pyridinyl, pyrido[3,2-d]pyrimidinyl,
pyrido[3,4-d]pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl,
pyrrolyl, quinazolinyl, quinoxalinyl, quinolinyl, isoquinolinyl,
tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl,
5,6,7,8-tetrahydrobenzo [4,5] thieno [2,3-d]pyrimdinyl,
6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno [2,3-d]pyrimidinyl,
5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl, thiazolyl,
thiadiazolyl, thiapyranyl, triazolyl, tetrazolyl, triazinyl,
thieno[2,3-d]pyrimidinyl, thieno[3,2-d]pyrimidinyl, thieno
[2,3-c]pridinyl, and thiophenyl (i.e., thienyl). Unless stated
otherwise in the specification, a heteroaryl moiety can be
optionally substituted by one or more substituents which
independently include: acyl, alkyl, alkenyl, alkynyl, alkoxy,
alkylaryl, cycloalkyl, aralkyl, aryl, aryloxy, amino, amido,
amidino, imino, azide, carbonate, carbamate, carbonyl, heteroalkyl,
heteroaryl, heteroarylalkyl, heterocycloalkyl, hydroxy, cyano,
halo, haloalkoxy, haloalkyl, ester, ether, mercapto, thio,
alkylthio, arylthio, thiocarbonyl, nitro, oxo, phosphate,
phosphonate, phosphinate, silyl, sulfinyl, sulfonyl, sulfonamidyl,
sulfoxyl, sulfonate, urea, --Si(R.sup.a).sub.3, --OR.sup.a,
--SR.sup.a, --OC(O)--R.sup.a, --N(R.sup.a).sub.2, --C(O)R.sub.a,
--C(O)OR.sup.a, --OC(O)N(R.sup.a).sub.2, --C(O)N(R.sup.a).sub.2,
--N(R.sup.a)C(O)OR.sup.a, --N(R.sup.a)C(O)R.sup.a,
--N(R.sup.a)C(O)N(R.sup.a).sub.2,
--N(R.sup.a)C(NR.sup.a)N(R.sup.a).sub.2,
--N(R.sup.a)S(O).sub.tN(R.sup.a).sub.2 (where t is 1 or 2),
--P(.dbd.O)(R.sup.a)(R.sup.a), or --O--P(.dbd.O)(OR.sup.a).sub.2
where each R.sup.a is independently hydrogen, alkyl, haloalkyl,
carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocycloalkyl,
heterocycloalkylalkyl, heteroaryl or heteroarylalkyl, and each of
these moieties can be optionally substituted as defined herein.
[0092] As used herein, the term "antibody" refers to molecules that
are capable of binding an epitope or antigenic determinant. The
term is meant to include whole antibodies and antigen-binding
fragments thereof, including single-chain antibodies. The
antibodies can be from any animal origin. Preferably, the
antibodies are mammalian, e.g., human, murine, rabbit, goat, guinea
pig, camel, horse and the like, or other suitable animals.
Antibodies may recognize polypeptide or polynucleotide antigens.
The term includes active fragments, including for example, an
antigen binding fragment of an immunoglobulin, a variable and/or
constant region of a heavy chain, a variable and/or constant region
of a light chain, a complementarity determining region (cdr), and a
framework region. The terms include polyclonal and monoclonal
antibody preparations, as well as preparations including hybrid
antibodies, altered antibodies, chimeric antibodies, hybrid
antibody molecules, F(ab).sub.2 and F(ab) fragments; Fv molecules
(for example, noncovalent heterodimers), dimeric and trimeric
antibody fragment constructs; minibodies, humanized antibody
molecules, and any functional fragments obtained from such
molecules, wherein such fragments retain specific binding.
[0093] As used herein, the term "epitope" refers to basic element
or smallest unit of recognition by an individual antibody or T-cell
receptor, and thus the particular domain, region or molecular
structure to which said antibody or T-cell receptor binds. An
antigen may consist of numerous epitopes while a hapten, typically,
may possess few epitopes.
[0094] As used herein, the term "immune system" diseases or
conditions refers to a group of conditions characterized by a
dysfunctioning immune system. These disorders can be characterized
in several different ways: by the component(s) of the immune system
affected, by whether the immune system is overactive or
underactive, or by whether the condition is congenital or acquired.
Autoimmune diseases or conditions are among immune system diseases
or conditions.
[0095] As used herein, the term "inflammatory" diseases or
conditions refers to a group of conditions including, rheumatoid
arthritis, osteoarthritis, juvenile idiopathic arthritis,
psoriasis, allergic airway disease (e.g., asthma, rhinitis),
inflammatory bowel diseases (e.g., Crohn's disease, colitis),
endotoxin-driven disease states (e.g., complications after bypass
surgery or chronic endotoxin states contributing to e.g. chronic
cardiac failure), and related diseases involving cartilage, such as
that of the joints.
[0096] As used herein, the term "autoimmune" diseases or conditions
refers to conditions arising from an abnormal immune response to a
normal body part. Examples of include, but not limited to
rheumatoid arthritis, lupus, Alzheimer's disease, multiple
sclerosis, Parkinson's disease, inflammatory bowel disease, and
psoriasis.
[0097] As used herein, the term "cancer" refers to or describes the
physiological condition in mammals that is typically characterized
by unregulated cell growth. Examples of cancer include but are not
limited to, carcinoma, lymphoma, sarcoma, blastoma and leukemia.
More particular examples of such cancers include squamous cell
carcinoma, lung cancer, pancreatic cancer, cervical cancer, bladder
cancer, hepatoma, breast cancer, colon carcinoma, and head and neck
cancer.
[0098] As used herein, the term "tumor" refers to any malignant or
neoplastic cell.
[0099] As used herein, the terms "polypeptide" and "protein" are
used interchangeably to refer to a polymer of amino acid residues,
and are not limited to a minimum length. Thus, peptides,
oligopeptides, dimers, multimers, and the like, are included within
the definition. Both full-length proteins and fragments thereof are
encompassed by the definition. The terms also include
post-expression modifications of the polypeptide, for example,
glycosylation, acetylation, phosphorylation, and the like.
Furthermore, a "polypeptide" may refer to a protein which includes
modifications, such as deletions, additions, and substitutions
(generally conservative in nature), to the native sequence, as long
as the protein maintains the desired activity. These modifications
may be deliberate or may be accidental.
[0100] As used herein, the term "sample" refers to a sample from a
human, animal, or to a research sample, e.g., a cell, tissue,
organ, fluid, gas, aerosol, slurry, colloid, or coagulated
material. The "sample" may be tested in vivo, e.g., without removal
from the human or animal, or it may be tested in vitro. The sample
may be tested after processing, e.g., by histological methods.
"Sample" also refers, e.g., to a cell comprising a fluid or tissue
sample or a cell separated from a fluid or tissue sample. "Sample"
may also refer to a cell, tissue, organ, or fluid that is freshly
taken from a human or animal, or to a cell, tissue, organ, or fluid
that is processed or stored.
[0101] As used herein, the term an "isolated" or "substantially
isolated" molecule (such as a polypeptide or polynucleotide) is one
that has been manipulated to exist in a higher concentration than
in nature or has been removed from its native environment. For
example, a subject antibody is isolated, purified, substantially
isolated, or substantially purified when at least 10%, or 20%, or
40%, or 50%, or 70%, or 90% of non-subject-antibody materials with
which it is associated in nature have been removed. For example, a
polynucleotide or a polypeptide naturally present in a living
animal is not "isolated," but the same polynucleotide or
polypeptide separated from the coexisting materials of its natural
state is "isolated." Further, recombinant DNA molecules contained
in a vector are considered isolated for the purposes of the present
invention. Isolated RNA molecules include in vivo or in vitro RNA
replication products of DNA and RNA molecules. Isolated nucleic
acid molecules further include synthetically produced molecules.
Additionally, vector molecules contained in recombinant host cells
are also isolated. Thus, not all "isolated" molecules need be
"purified."
[0102] As used herein, the term "purified" when used in reference
to a molecule, it means that the concentration of the molecule
being purified has been increased relative to molecules associated
with it in its natural environment, or environment in which it was
produced, found or synthesized. Naturally associated molecules
include proteins, nucleic acids, lipids and sugars but generally do
not include water, buffers, and reagents added to maintain the
integrity or facilitate the purification of the molecule being
purified. According to this definition, a substance may be 5% or
more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or
more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or
more, 98% or more, 99% or more, or 100% pure when considered
relative to its contaminants.
DETAILED DESCRIPTION OF THE INVENTION
[0103] The invention provides novel, orally available, potent and
selective inhibitors or inactivators of PADs, pharmaceutical
compositions and methods of use thereof. The compounds and
pharmaceutical compositions of the invention may be used to treat
immune system disorders and inflammatory diseases and conditions
(e.g., rheumatoid arthritis, lupus). Diseases and conditions that
may benefit from treatment using the compounds and pharmaceutical
compositions of the invention include ulcerative colitis, spinal
cord injury, breast cancer, and atherosclerosis.
[0104] Certain benzimidazole-based PAD inhibitors or inactivators
disclosed herein display improved metabolic stability, cell
permeability and/or potency. It is believed that certain compounds
of the invention can access binding regions within PAD2 and PAD4
that had not been previously accessed.
[0105] For example, certain compounds disclosed herein possess
superior PAD2 inhibition (k.sub.inact/K.sub.I>300,000
k.sub.inact/K.sub.I) and selectivity (.about.100-fold selective for
PAD2 vs PAD4). Certain compounds disclosed herein exhibit improved
cell permeability (e.g., over a 10-fold increase in Clog P compared
to the peptide-based inhibitor Cl-amidine).
[0106] The invention also provides novel potent and selective
molecular probes (e.g., imaging probes) for PADs based on the
inhibitors or inactivators of PADs disclosed herein in conjugation
with detectable labels such as fluorescent dyes, and methods of use
thereof. The molecular imaging probes of the invention may be used
to screen compounds for PAD inhibition or inactivation.
[0107] Aberrantly upregulated protein citrullination is associated
with a variety of autoimmune diseases (e.g., rheumatoid arthritis,
multiple sclerosis, lupus, and ulcerative colitis), as well as
certain cancers. Given these disease links, the protein arginine
deiminases have garnered significant recent interest. (Jones, et
al. 2009 Curr Opin Drug Discov Devel 12, 616-627; Bicker, et al.
2013 Biopolymers 99, 155-163; Vossenaar, et al. 2003 Bioessays 25,
1106-1118.)
[0108] The most deeply investigated disease associated with
aberrantly increased PAD activity is RA, where these patients
produce autoantibodies that target numerous citrullinated proteins
(e.g., citrullinated keratin, fibrin, vimentin and enolase).
Importantly, the presence of these autoantibodies is the most
specific diagnostic test available for RA. The presence of these
anti-citrullinated protein antibodies (i.e., ACPA) is highly
predictive of both disease incidence and severity. (Van Steendam,
et al. 2011 Rheumatology (Oxford) 50, 830-837; Puszczewicz, et al.
2011 Arch Med Sci 7, 189-194; van Boekel, et al. 2002 Arthritis Res
4, 87-93; Masson-Bessiere, et al. 2001J Immunol 166, 4177-4184;
Burska, et al. 2014 Mediators Inflamm 2014, 492873.)
[0109] In addition to ACPA, the PADs themselves are present in the
synovial joints of patients with RA where they remain active and
citrullinate proteins within the joints that bind ACPA, thereby
setting up a classic positive feedback loop that recruits
additional immune cells into the joint, the release of additional
PAD isozymes into the synovium and enhanced protein citrullination
and consequent inflammation. (Burska, et al. 2014 Mediators Inflamm
2014, 492873; Damgaard, et al. 2014 Arthritis Res Ther 16,
498.)
[0110] Without wishing to be bound by the theories discussed
herein, the specific cells that release PAD isozymes into the
joints of RA patients are likely neutrophils. Neutrophils are the
predominant white blood cell in humans and are generally the first
responders to signs of infection/inflammation. Depending on the
environmental cues, a subset of these cells will undergo a novel
form of cell death known as Neutrophil Extracellular trap formation
(NET) or NETosis. Neutrophils have long been known to be important
players in the development and progression of RA as they are the
predominant cell type in the synovial fluid of RA patients.
Enhanced NETosis, as is observed in RA, also results in the
exposure of citrullinated autoantigens, which is key to the
progression of RA. (Khandpur, et al. 2013 Sci Transl Med 5,
178ra140; Li, et al. 2010 J Exp Med 207, 1853-1862; Brinkmann, et
al. 2004 Science 303, 1532-1535; Ottonello, et al. 2002
Rheumatology (Oxford) 41, 1249-1260; Weissmann, et al. 1984
Inflammation 8 Suppl, 53-14.)
[0111] In regard to characteristic features of other inflammatory
diseases, enhanced citrullination in the inflamed regions indicates
that abberant NETosis may contribute to these diseases as well. In
addition, since the PADs are histone-modifying enzymes that
contribute to the epigenetic control of gene expression, there is
emerging evidence to suggest that enhanced PAD activity promotes an
inflamed state by altering the expression and/or activity of key
cytokines and chemokines. (Kawalkowska, et al. 2016 Sci Rep 6,
26430; Loos, et al. 2008 Blood 112, 2648-2656; Proost, et al. 2008
J Exp Med 205, 2085-2097; Struyf, et al. 2009 J Immunol 182,
666-674.)
[0112] The role the PADs play in these diseases is further
highlighted by the efficacy of several PAD inhibitors in a variety
of pre-clinical disease models. Specifically, the first-generation
irreversible inhibitor Cl-amidine has demonstrated efficacy in
animal models of rheumatoid arthritis, lupus, ulcerative colitis,
spinal cord injury, breast cancer, and atherosclerosis. (Khandpur,
et al. 2013 Sci Transl Med 5, 178ra140; Wang, et al. 2012 J Biol
Chem 287, 25941-25953; Chumanevich, et al. 2011 Am J Physiol
Gastrointest Liver Physiol 300, G929-938; Lange, et al. 2011 Dev
Biol 355, 205-214; Causey, et al. 2011 J Med Chem 54, 6919-6935;
Knight, et al. 2012 Curr Opin Rheumatol 24, 441-450; Knight, et al.
2013 J Clin Invest 123, 2981-2993; Smith, et al. 2014 Arthritis
Rheumatol 66, 2532-2544; Knight, et al. 2014 Circ Res 114, 947-956;
McElwee, et al. 2012 BMC Cancer 12, 500.)
[0113] The first-generation inhibitors suffer a number of
limitations including their susceptibility to proteolysis and poor
membrane permeability. This prompted the development of
second-generation inhibitors predicated on improving metabolic
stability and membrane permeability.
##STR00005##
[0114] Reports have shown that a second-generation inhibitor,
BB-Cl-amidine, exhibits enhanced efficacy over Cl-amidine in animal
models of lupus and RA. Moreover, the allosteric inhibitor GSK199
also shows efficacy in an RA model. Together, these findings have
validated the PADs as viable therapeutic targets for a wide range
of inflammatory conditions. (Kawalkowska, et al. 2016 Sci Rep 6,
26430; Knight, et al. 2015 Ann Rheum Dis 74, 2199-2206; Ghari, et
al. 2016 Sci Adv 2, e1501257; Willis, V. C. The Role of
Citrullination in the Development of Mouse and Human Inflammatory
Arthritis. University of Colorado Boulder, Boulder, Colo.,
2012.)
[0115] The novel PAD inhibitors or inactivators disclosed herein
may be used to treat a variety of diseases where PAD activity is
dysregulated, for example, RA, lupus, atherosclerosis as well as
other inflammatory diseases. These PAD inhibitors may also find use
in treating individuals with spinal cord injuries as well as
psoriasis. Specifically, certain PAD inhibitors disclosed herein
exhibit enhanced potency and selectivity for PAD2, and so they may
be used to treat diseases where PAD2 activity is upregulated.
Certain disclosed inhibitors may be useful in treating certain
cancers such as HER2 overexpressing breast cancers and certain lung
cancers, as well as in the treatment of multiple sclerosis.
[0116] The PAD molecular probes disclosed herein may be
specifically designed with a terminal alkyne so they can covalently
modify the PAD enzyme and undergo a subsequent "click" reaction
with either TAMRA-N.sub.3 or Biotin-N.sub.3. These probes may find
use in identifying particular diseases where the PADs are
upregulated, as well as identifying PAD isozyme-specific diseases.
These probes should also find utility in identifying proteins that
interact with the PADs as well as in identifying compounds with
inhibitory properties or binding affinities to PADs.
[0117] In one aspect, the invention generally relates to a compound
having the structural formula (I),
##STR00006##
wherein
[0118] each of R.sub.a and R.sub.b is independently selected from
the group consisting of H, D and F;
[0119] L is a bivalent hydrocarbyl linker, optionally with one or
more carbon atoms replaced by a heteroatom selected from the group
consisting of O, S and N;
[0120] X is a halogen atom;
[0121] Y is N, O or S; provided that when Y is S or O, its bonding
to the adjacent carbons are single bonds;
[0122] Z is N--R.sub.1, O or S;
[0123] R.sub.1 is selected from the group consisting of: H, a
C.sub.1-6 alkyl, OH, a C.sub.1-3 alkoxy, CF.sub.3, COCH.sub.3, and
COCF.sub.3 groups;
[0124] each of R.sub.2, R.sub.3, R.sub.4 and R.sub.5 is
independently selected from the group consisting of: H, hydroxyl,
halogen atom, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, alkynyl,
CF.sub.2R.sub.c, and OCF.sub.2R.sub.c groups, where R.sub.c is H, F
or alkyl; and
[0125] R.sub.6 is a group comprising a cyclic alkyl or aryl
moiety,
or a pharmaceutically acceptable form thereof.
[0126] In certain embodiments, each of R.sub.a and R.sub.b is
H.
[0127] In certain embodiments, L is a --(CH.sub.2).sub.n--, wherein
n is an integer from 1 to about 4 (e.g., n is 1, 2, 3, or 4). In
preferred certain embodiments, n is 3 and L is
--(CH.sub.2).sub.3--.
[0128] In certain embodiments, Y is N and Z is N--R.sub.1 with the
compound having the structural formula (II):
##STR00007##
[0129] In certain embodiments, X is F. In certain embodiments, X is
Cl.
[0130] In certain embodiments, R.sub.1 is a C.sub.1-6 alkyl group.
In certain embodiments, R.sub.1 is H.
[0131] In certain embodiments, each of R.sub.2, R.sub.3, R.sub.4
and R.sub.5 is H.
[0132] In certain embodiments, at least one of R.sub.2, R.sub.3,
R.sub.4 and R.sub.5 is a group selected from the group consisting
of hydroxyl, F, Cl, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, alkynyl,
CF.sub.2R.sub.c, and OCF.sub.2R.sub.c groups, where R.sub.c is H, F
or alkyl.
[0133] In certain embodiments, one of R.sub.2 and R.sub.3 is a
group selected from the group consisting of hydroxyl, F, Cl,
C.sub.1-6 alkyl, C.sub.1-6 alkoxy, alkynyl, CF.sub.2R.sub.c, and
OCF.sub.2R.sub.c groups, where R.sub.c is H, F or alkyl.
[0134] In certain embodiments, R.sub.6 is a cyclic, nonaromatic,
hydrocarbyl group.
[0135] In certain embodiments, R.sub.6 is a cyclic, aromatic,
hydrocarbyl group.
[0136] In certain embodiments, R.sub.6 is a heterocyclic,
nonaromatic, hydrocarbyl group.
[0137] In certain embodiments, R.sub.6 is a heterocyclic, aromatic,
hydrocarbyl group.
[0138] In certain embodiments, R.sub.6 is selected from the group
consisting of:
##STR00008##
wherein
[0139] R.sub.7 is independently selected from the group consisting
of H, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, hydroxyl, halogen atoms,
aryl, --COOR.sub.8, ethynyl, alkynyl, CF.sub.2R.sub.c, and
OCF.sub.2R.sub.c, where RC is H, F or alkyl;
[0140] R.sub.8 is independently selected from the group consisting
of H and C.sub.1-6 alkyl; and
[0141] R.sub.9 is CH.sub.2, O, NH.sub.2 or S.
[0142] In certain embodiments, R.sub.7 is H.
[0143] In certain embodiments, R.sub.8 is a C.sub.1-6 alkyl
group.
[0144] In another aspect, the invention generally relates to a
pharmaceutical composition comprising a compound having the
structural formula of (I):
##STR00009##
wherein,
[0145] each of R.sub.a and R.sub.b is independently selected from
the group consisting of H, D and F;
[0146] L is a bivalent hydrocarbyl linker, optionally with one or
more carbon atoms replaced by a heteroatom selected from the group
consisting of O, S and N;
[0147] X is a halogen atom;
[0148] Y is N, O or S; provided that when Y is S or O, its bonding
to the adjacent carbons are single bonds;
[0149] Z is N--R.sub.1, O or S;
[0150] R.sub.1 is selected from the group consisting of: H, a
C.sub.1-6 alkyl, OH, a C.sub.1-3 alkoxy, CF.sub.3, COCH.sub.3, and
COCF.sub.3 groups;
[0151] each of R.sub.2, R.sub.3, R.sub.4 and R.sub.5 is
independently selected from the group consisting of: H, hydroxyl,
halogen atom, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, alkynyl,
CF.sub.2R.sub.c, and OCF.sub.2R.sub.c groups, where R.sub.c is H, F
or alkyl; and
[0152] R.sub.6 is a group comprising a cyclic alkyl or aryl
moiety,
or a pharmaceutically acceptable form thereof, effective to treat,
prevent, or reduce one or more diseases or disorders, in a mammal,
including a human, and a pharmaceutically acceptable excipient,
carrier, or diluent.
[0153] In certain embodiments, the one or more diseases or
conditions are selected from the group consisting of immune system
diseases or disorders, inflammatory diseases or disorders, and
cancer or related diseases or disorders.
[0154] In certain embodiments, the one or more diseases or
conditions are selected from autoimmune diseases or disorders.
[0155] In certain embodiments, the one or more diseases or
conditions are selected from the group consisting of lupus,
rheumatoid arthritis, Alzheimer's disease, multiple sclerosis and
Parkinson's disease.
[0156] In certain embodiments, the disease or condition is
lupus.
[0157] In certain embodiments, the disease or condition is
rheumatoid arthritis.
[0158] Pharmaceutical compositions of the invention includes that
of a compound of the invention disclosed herein.
[0159] In yet another aspect, the invention generally relates to a
unit dosage form comprising a pharmaceutical composition disclosed
herein.
[0160] In yet another aspect, the invention generally relates to a
method for treating, reducing, or preventing a disease or disorder.
The method includes administering to a subject in need thereof a
pharmaceutical composition comprising a compound having the
structural formula of (I):
##STR00010##
wherein,
[0161] each of R.sub.a and R.sub.b is independently selected from
the group consisting of H, D and F;
[0162] L is a bivalent hydrocarbyl linker, optionally with one or
more carbon atoms replaced by a heteroatom selected from the group
consisting of O, S and N;
[0163] X is a halogen atom;
[0164] Y is N, O or S; provided that when Y is S or O, its bonding
to the adjacent carbons are single bonds;
[0165] Z is N--R.sub.1, O or S;
[0166] R.sub.1 is selected from the group consisting of: H, a
C.sub.1-6 alkyl, OH, a C.sub.1-3 alkoxy, CF.sub.3, COCH.sub.3, and
COCF.sub.3 groups;
[0167] each of R.sub.2, R.sub.3, R.sub.4 and R.sub.5 is
independently selected from the group consisting of: H, hydroxyl,
halogen atom, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, alkynyl,
CF.sub.2R.sub.c, and OCF.sub.2R.sub.c groups, where R.sub.c is H, F
or alkyl; and
[0168] R.sub.6 is a group comprising a cyclic alkyl or aryl
moiety,
or a pharmaceutically acceptable form thereof, and a
pharmaceutically acceptable excipient, carrier, or diluent,
effective to treat, prevent, or reduce one or more diseases or
disorders, in a mammal, including a human.
[0169] In certain embodiments, the one or more diseases or
conditions are selected from the group consisting of immune system
diseases or disorders, inflammatory diseases or disorders, and
cancer or related diseases or disorders.
[0170] In certain embodiments, the one or more diseases or
conditions are selected from autoimmune diseases or disorders
[0171] In certain embodiments, the one or more diseases or
conditions are selected from the group consisting of lupus,
rheumatoid arthritis, Alzheimer's disease, multiple sclerosis and
Parkinson's disease.
[0172] In certain embodiments, the disease or condition is
lupus.
[0173] In certain embodiments, the disease or condition is
rheumatoid arthritis.
[0174] In yet another aspect, the invention generally relates to a
method for inhibiting or inactivating a protein arginine deiminase,
comprising administering to a subject in need thereof a
pharmaceutical composition comprising a compound having the
structural formula of (I):
##STR00011##
wherein,
[0175] each of R.sub.a and R.sub.b is independently selected from
the group consisting of H, D and F;
[0176] L is a bivalent hydrocarbyl linker, optionally with one or
more carbon atoms replaced by a heteroatom selected from the group
consisting of O, S and N;
[0177] X is a halogen atom;
[0178] Y is N, O or S; provided that when Y is S or O, its bonding
to the adjacent carbons are single bonds;
[0179] Z is N--R.sub.1, O or S;
[0180] R.sub.1 is selected from the group consisting of: H, a
C.sub.1-6 alkyl, OH, a C.sub.1-3 alkoxy, CF.sub.3, COCH.sub.3, and
COCF.sub.3 groups;
[0181] each of R.sub.2, R.sub.3, R.sub.4 and R.sub.5 is
independently selected from the group consisting of: H, hydroxyl,
halogen atom, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, alkynyl,
CF.sub.2R.sub.c and OCF.sub.2R.sub.c groups, where R.sub.c is H, F
or alkyl; and
[0182] R.sub.6 is a group comprising a cyclic alkyl or aryl
moiety,
or a pharmaceutically acceptable form thereof, and a
pharmaceutically acceptable excipient, carrier, or diluent,
effective to inhibit or inactivate a biological function of a
protein arginine deiminase, in a mammal, including a human.
[0183] In certain embodiments, the protein arginine deiminase is
selected from the group consisting of: PAD1, PAD2, PAD3 and
PAD4.
[0184] In certain embodiments, the protein arginine deiminase is
PAD2.
[0185] In certain embodiments, the protein arginine deiminase is
PAD4.
[0186] In yet another aspect, the invention generally relates to a
molecular imaging probe having the structural formula:
A.sub.F-L.sub.F-W (II)
wherein
[0187] A.sub.F is a group comprising an optically detectable
moiety;
[0188] L.sub.F is a linking group; and
[0189] W is group comprising a benzimidazole moiety, or a
derivative or analog thereof, capable of inhibiting or inactivating
a biological function of a protein arginine deiminase.
[0190] In certain embodiments, A.sub.F is a group comprising a
fluorophore.
[0191] In certain embodiments, the fluorophore is selected from the
group consisting of xanthene dyes, cyanine dyes, coumarin dyes and
bodipy dyes.
[0192] In certain embodiments, the fluorophore is a xanthene dye
selected from the group consisting of fluorescein, eosins, and
rhodamines.
[0193] In certain embodiments, the fluorophore is a cyanine
dye.
[0194] In certain embodiments, the fluorophore is a coumarin
dye.
[0195] In certain embodiments, the fluorophore is a bodipy dye.
[0196] In certain embodiments, the protein arginine deiminase is
selected from the group consisting of: PAD1, PAD2, PAD3 and
PAD4.
[0197] In certain embodiments, the protein arginine deiminase is
PAD2.
[0198] In certain embodiments, the protein arginine deiminase is
PAD4.
[0199] In certain embodiments, W is a monovalent radical derived
from a compound having the structural formula:
##STR00012##
wherein,
[0200] each of R.sub.a and R.sub.b is independently selected from
the group consisting of H, D and F;
[0201] L is a bivalent hydrocarbyl linker, optionally with one or
more carbon atoms replaced by a heteroatom selected from the group
consisting of O, S and N;
[0202] X is a halogen atom;
[0203] Y is N, O or S; provided that when Y is S or O, its bonding
to the adjacent carbons are single bonds;
[0204] Z is N--R.sub.1, O or S;
[0205] R.sub.1 is selected from the group consisting of: H, a
C.sub.1-6 alkyl, OH, a C.sub.1-3 alkoxy, CF.sub.3, COCH.sub.3, and
COCF.sub.3 groups;
[0206] each of R.sub.2, R.sub.3, R.sub.4 and R.sub.5 is
independently selected from the group consisting of: H, hydroxyl,
halogen atom, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, alkynyl,
CF.sub.2R.sub.c and OCF.sub.2R.sub.c groups, where R.sub.c is H, F
or alkyl; and
[0207] R.sub.6 is a group comprising a cyclic alkyl or aryl
moiety.
[0208] In yet another aspect, the invention generally relates to a
method for identifying a protein arginine deiminase inhibitor or
inactivator. The method includes: performing a competitive assay
wherein a test compound competes with a molecular imaging probe
disclosed herein to bind to a protein arginine deiminase; and
measuring fluorescence to determine an amount of fluorescent
protein arginine deiminase present in the test assay.
[0209] In certain embodiments, the method further includes:
performing a control assay wherein the molecular imaging probe
binds to the protein arginine deiminase; and measuring fluorescence
to determine an amount of fluorescent protein arginine deiminase
present in the control assay.
[0210] In certain embodiments, a change in fluorescence in the
assay greater than a pre-selected value when compared to the
control assay is indicative that the test compound is an inhibitor
to the protein arginine deiminase.
[0211] In certain embodiments, the change in fluorescence in the
assay is a decrease in fluorescence in the assay.
EXAMPLES
##STR00013##
TABLE-US-00001 [0212] TABLE 1a k.sub.inact/K.sub.I values for
compounds 5a-w PAD1 k.sub.inact/K.sub.I PAD2 k.sub.inact/K.sub.I
PAD3 k.sub.inact/K.sub.I PAD4 k.sub.inact/K.sub.I Compound
(M.sup.-1min.sup.-1) (M.sup.-1min.sup.-1) (M.sup.-1min.sup.-1)
(M.sup.-1min.sup.-1) 5a (X = F) 4500 (.+-.300).sup.a 475
(.+-.20).sup.a 900 (.+-.200).sup.a 8500 (.+-.1300).sup.a 5b (X =
Cl) 7950 (.+-.675).sup.a 500 (.+-.50).sup.a 9770 (.+-.350).sup.a
15250 (.+-.675).sup.a 5c (X = F) 1500 (.+-.400).sup.a 785
(.+-.60).sup.a 2800 (.+-.500).sup.a 6500 (.+-.700).sup.a 5d (X =
Cl) 2800 (.+-.550).sup.a 4275 (.+-.250).sup.a 9875 (.+-.500).sup.a
10000 (.+-.1000).sup.a 5e (R.sub.1 = R.sub.2 = R.sub.3 = R.sub.4 =
R.sub.5 = H, 900 (.+-.100).sup.a 1200 (.+-.30).sup.a 3400
(.+-.140).sup.a 3750 (.+-.250).sup.a X = F) 5f (R.sub.1 = R.sub.2 =
R.sub.3 = R.sub.4 = R.sub.5 = H, 16100 (.+-.1500).sup.a 4100
(.+-.400).sup.a 6800 (.+-.200).sup.a 13300 (.+-.3000).sup.a X = Cl)
5g (R.sub.1 = Me, 1280 (.+-.120).sup.a 820 (.+-.90).sup.b 1600
(.+-.210).sup.a 450 (.+-.50).sup.b R.sub.2 = R.sub.3 = R.sub.4 =
R.sub.5 = H, X = F) 5h (R.sub.1 = Me, R.sub.2 = H, 6750
(.+-.500).sup.a 2430 (.+-.400).sup.a 6020 (.+-.650).sup.a 3110
(.+-.420).sup.a R.sub.2 = R.sub.3 = R.sub.4 = R.sub.5 = H, X = Cl)
5i (R.sub.1 = Et, R.sub.2 = H, 760 (.+-.100).sup.a 1160
(.+-.100).sup.b 1800 (.+-.210).sup.a 30 (.+-.5).sup.b R.sub.2 =
R.sub.3 = R.sub.4 = R.sub.5 = H, X = F) 5j (R.sub.1 = Et, R.sub.2 =
H, 5060 (.+-.650).sup.a 4850 (.+-.320).sup.a 17040 (.+-.2100).sup.a
4150 (.+-.420).sup.a R.sub.2 = R.sub.3 = R.sub.4 = R.sub.5 = H, X =
Cl) 5k (R.sub.1 = .sup.iPr, R.sub.2 = H, 20 (.+-.10).sup.a 680
(.+-.50).sup.b 1700 (.+-.190).sup.a 20 (.+-.5).sup.b R.sub.2 =
R.sub.3 = R.sub.4 = R.sub.5 = H, X = F) 5l (R.sub.1 = .sup.iPr,
R.sub.2 = H, 1220 (.+-.190).sup.a 1490 (.+-.180).sup.a 11500
(.+-.1500).sup.a 950 (.+-.130).sup.a R.sub.2 = R.sub.3 = R.sub.4 =
R.sub.5 = H, X = Cl) 5m (R.sub.1 = Me, 1870 (.+-.180).sup.a 7920
(.+-.1100).sup.a 2900 (.+-.310).sup.a 790 (.+-.95).sup.a R.sub.2 =
R.sub.3 = R.sub.4 = R.sub.5 = H, R.sub.2 = OMe, X = F) 5n (R.sub.1
= Me, 34800 (.+-.4200).sup.a 27800 (.+-.3500).sup.a 5500
(.+-.810).sup.a 7900 (.+-.710).sup.a R.sub.2 = R.sub.3 = R.sub.4 =
R.sub.5 = H, R.sub.2 = OMe, X = Cl) 5o (R.sub.1 = R.sub.2 = R.sub.3
= R.sub.4 = H, 1400 (.+-.120).sup.a 1700 (.+-.210).sup.a 1620
(.+-.190).sup.a 3200 (.+-.380).sup.a R.sub.5 = F, X = F) 5p
(R.sub.1 = R.sub.2 = R.sub.3 = R.sub.4 = H, 20600 (.+-.2400).sup.a
5500 (.+-.620).sup.a 10530 (.+-.1100).sup.a 16800 (.+-.1800).sup.a
R.sub.5 = F, X = Cl) 5q (R.sub.1 = R.sub.2 = R.sub.5 = H, 1860
(.+-.190).sup.a 1570 (.+-.200).sup.a 1530 (.+-.140).sup.a 3600
(.+-.420).sup.a R.sub.3 = R.sub.4 = F, X = F) 5r (R.sub.1 = R.sub.2
= R.sub.5 = H, 27400 (.+-.3100).sup.a 5600 (.+-.610).sup.a 14360
(.+-.1500).sup.a 20220 (.+-.2150).sup.a R.sub.3 = R.sub.4 = F, X =
Cl) 5s (R.sub.1 = R.sub.2 = H, R.sub.3 = R.sub.4 = 1790
(.+-.230).sup.a 1420 (.+-.170).sup.a 1100 (.+-.160).sup.a 4040
(.+-.480).sup.a R.sub.5 = F, X = F) 5t (R.sub.1 = R.sub.2 = H,
R.sub.3 = R.sub.4 = 30480 (.+-.5350).sup.a 5730 (.+-.590).sup.a
7180 (.+-.790).sup.a 21250 (.+-.2500).sup.a R.sub.5 = F, X = Cl) 5u
(R.sub.1 = R.sub.2 = R.sub.3 = R.sub.4 = H, 4650 (.+-.80).sup.a 975
(.+-.30).sup.a 1025 (.+-.25).sup.a 1770 (.+-.110).sup.a R.sub.5 =
CCH, X = F) 5v (R.sub.1 = R.sub.2 = R.sub.3 = R.sub.4 = H, 415
(.+-.70).sup.a 875 (.+-.70).sup.a 375 (.+-.50).sup.a 1300
(.+-.100).sup.a R.sub.5 = CCH, X = Cl) .sup.aA single k.sub.obs was
determined. .sup.bk.sub.inact/K.sub.I was determined from a linear
fit.
TABLE-US-00002 TABLE 1b Summary of isozyme selectivity for
compounds 5a-w Fold Fold Fold Fold PAD1 PAD2 PAD3 PAD4 Compound
Selectivity Selectivity Selectivity Selectivity 5a (X = F) 9.5 1.0
1.9 18 5b (X = Cl) 16 1.0 20 31 5c (X = F) 1.9 1.0 3.6 8.3 5d (X =
Cl) 1.0 1.5 3.5 3.6 5e (R.sub.1 = R.sub.2 = R.sub.3 = R.sub.4 = 1.0
1.3 3.8 4.2 R.sub.5 = H, X = F) 5f (R.sub.1 = R.sub.2 = R.sub.3 =
R.sub.4 = 3.9 1.0 1.7 3.2 R.sub.5 = H, X = Cl) 5g (R.sub.1 = Me,
R.sub.2 = R.sub.3 = 2.8 1.8 3.6 1.0 R.sub.4 = R.sub.5 = H, X = F)
5h (R.sub.1 = Me, R.sub.2 = H, 2.8 1.0 2.5 1.3 R.sub.2 = R.sub.3 =
R.sub.4 = R.sub.5 = H, X = Cl) 5i (R.sub.1 = Et, R.sub.2 = H, 25 39
60 1.0 R.sub.2 = R.sub.3 = R.sub.4 = R.sub.5 = H, X = F) 5j
(R.sub.1 = Et, R.sub.2 = H, 1.2 1.2 4.1 1.0 R.sub.2 = R.sub.3 =
R.sub.4 = R.sub.5 = H, X = Cl) 5k (R.sub.1 = .sup.iPr, R.sub.2 = H,
1.0 34 85 1.0 R.sub.2 = R.sub.3 = R.sub.4 = R.sub.5 = H, X = F) 5l
(R.sub.1 = .sup.iPr, R.sub.2 = H, 1.3 1.6 12 1.0 R.sub.2 = R.sub.3
= R.sub.4 = R.sub.5 = H, X = Cl) 5m (R.sub.1 = Me, R.sub.2 =
R.sub.3 = 2.4 10 3.7 1.0 R.sub.4 = R.sub.5 = H, R.sub.2 = OMe, X =
F) 5n (R.sub.1 = Me, R.sub.2 = R.sub.3 = 6.3 5.1 1.0 1.4 R.sub.4 =
R.sub.5 = H, R.sub.2 = OMe, X = Cl) 5o (R.sub.1 = R.sub.2 = R.sub.3
= R.sub.4 = 1.0 1.2 1.2 2.3 H, R.sub.5 = F, X = F) 5p (R.sub.1 =
R.sub.2 = R.sub.3 = R.sub.4 = 3.7 1.0 1.9 3.1 H, R.sub.5 = F, X =
Cl) 5q (R.sub.1 = R.sub.2 = R.sub.5 = H, 1.2 1.0 1.0 2.4 R.sub.3 =
R.sub.4 = F, X = F) 5r (R.sub.1 = R.sub.2 = R.sub.5 = H, 4.9 1.0
2.6 3.6 R.sub.3 = R.sub.4 = F, X = Cl) 5s (R.sub.1 = R.sub.2 = H
R.sub.3 = 1.6 1.3 1.0 3.7 R.sub.4 = R5 = F, X = F) 5t (R.sub.1 =
R.sub.2 = H, R.sub.3 = 5.3 1.0 1.3 3.7 R.sub.4 = R.sub.5 = F, X =
Cl) 5u (R.sub.1 = R.sub.2 = R.sub.3 = R.sub.4 = 4.8 1.0 1.1 1.8 H,
R.sub.5 = CCH, X = F) 5v (R.sub.1 = R.sub.2 = R.sub.3 = R.sub.4 =
1.1 2.3 1.0 3.5 H, R.sub.5 = CCH, X = Cl)
##STR00014##
TABLE-US-00003 TABLE 2a k.sub.inact/K.sub.I values for compounds
6a-j PAD1 k.sub.inact/K.sub.I PAD2 k.sub.inact/K.sub.I PAD3
k.sub.inact/K.sub.I PAD4 k.sub.inact/K.sub.I Compound
(M.sup.-1min.sup.-1) (M.sup.-1min.sup.-1) (M.sup.-1min.sup.-1)
(M.sup.-1min.sup.-1) 6a (R.sub.1 = R.sub.2 = H, X = F) 16500
(.+-.2200).sup.a 1500 (.+-.300).sup.a 6500 (.+-.300).sup.a 24300
(.+-.1800).sup.a 6b (R.sub.1 = R.sub.2 = H, X = Cl) 28500
(.+-.2900).sup.a 3450 (.+-.100).sup.a 21250 (.+-.2750).sup.a 31000
(.+-.1550).sup.a 6c (R.sub.1 = Me, R.sub.2 = H, X = F) 32300
(.+-.4400).sup.a 11800 1030 (.+-.150).sup.a 800 (.+-.90).sup.b
(.+-.1090).sup.b 6d (R.sub.1 = Me, R.sub.2 = H, 32200
(.+-.3540).sup.a 14400 7500 (.+-.810).sup.a 7800 (.+-.770).sup.a X
= Cl) (.+-.1230).sup.a 6e (R.sub.1 = Et, R.sub.2 = H, X = F) 39000
(.+-.4600).sup.a 31600 1750 (.+-.180).sup.a 1010 (.+-.170).sup.b
(.+-.3030).sup.b 6f (R.sub.1 = Et, R.sub.2 = H, X = Cl) 47100
(.+-.4600).sup.a 17400 11000 (.+-.1300).sup.a 13700
(.+-.2100).sup.a (.+-.1500).sup.a 6g (R.sub.1 = .sup.iPr, R.sub.2 =
H, X = F) 22700 (.+-.2400).sup.a 24700 2010 (.+-.190).sup.a 460
(.+-.80).sup.a (.+-.2900).sup.a 6h (R.sub.1 = .sup.iPr, R.sub.2 =
H, 49000 (.+-.5100).sup.a 27100 2700 (.+-.290).sup.a 720
(.+-.90).sup.a X = Cl) (.+-.2400).sup.a 6i (R.sub.1 = Me, R.sub.2 =
OMe, 37600 (.+-.3900).sup.a 20400 6800 (.+-.770).sup.a 6040
(.+-.610).sup.a X = F) (.+-.1900).sup.a 6j (R.sub.1 = Me, R.sub.2 =
OMe, 67300 (.+-.7100).sup.a 18500 9600 (.+-.1200).sup.a 14100 X =
Cl) (.+-.1900).sup.a (.+-.1600).sup.a .sup.aA single k.sub.obs was
determined. .sup.bk.sub.inact/K.sub.I was determined from a linear
fit.
TABLE-US-00004 TABLE 2b Summary of isozyme selectivity for
compounds 6a-j Fold Fold Fold Fold PAD1 PAD2 PAD3 PAD4 Compound
Selectivity Selectivity Selectivity Selectivity 6a (R.sub.1 =
R.sub.2 = H, 11 1.0 4.3 16 X = F) 6b (R.sub.1 = R.sub.2 = H, 8.3
1.0 6.2 9.0 X = Cl) 6c (R.sub.1 = Me, R.sub.2 = H, 40 15 1.3 1.0 X
= F) 6d (R.sub.1 = Me, R.sub.2 = H, 4.3 1.9 1.0 1.0 X = Cl) 6e
(R.sub.1 = Et, R.sub.2 = H, 39 31 1.7 1.0 X = F) 6f (R.sub.1 = Et,
R.sub.2 = H, 4.3 1.6 1.0 1.2 X = Cl) 6g (R.sub.1 = .sup.iPr,
R.sub.2 = H, 49 54 4.4 1.0 X = F) 6h (R.sub.1 = .sup.iPr, R.sub.2 =
H, 68 38 3.8 1.0 X = Cl) 6i (R.sub.1 = Me, R.sub.2 = 6.2 3.4 1.1
1.0 OMe, X = F) 6j (R.sub.1 = Me, R2 = 7.0 1.9 1.0 1.5 OMe, X =
Cl)
##STR00015##
TABLE-US-00005 TABLE 3a k.sub.inact/K.sub.I values for compounds
7a-v PAD1 k.sub.inact/K.sub.I PAD2 k.sub.inact/K.sub.I PAD3
k.sub.inact/K.sub.I PAD4 k.sub.inact/K.sub.I Compound
(M.sup.-1min.sup.-1) (M.sup.-1min.sup.-1) (M.sup.-1min.sup.-1)
(M.sup.-1min.sup.-1) 7a (R.sub.1 = H, R.sub.2 = H, R.sub.3 = H,
77180 (.+-.15000).sup.a 10600 (.+-.3290).sup.c 3200 (.+-.450).sup.a
33100 X = F) (.+-.3350).sup.b 7b (R.sub.1 = H, R.sub.2 = H, R.sub.3
= H, 180830 65400 (.+-.9400).sup.a 28020 58100 X = Cl)
(.+-.23500).sup.a (.+-.3100).sup.a (.+-.4600).sup.a 7c (R.sub.1 =
Me, R.sub.2 = H, 68670 (.+-.9600).sup.a 24800 (.+-.3200).sup.a 2800
(.+-.310).sup.a 2910 (.+-.270).sup.a R.sub.3 = H, X = F) 7d
(R.sub.1 = Me, R.sub.2 = H, 146000 72900 (.+-.9350).sup.a 48500
23900 R.sub.3 = H, X = Cl) (.+-.22500).sup.a (.+-.3750).sup.a
(.+-.2760).sup.a 7e (R.sub.1 = Et, R.sub.2 = H, R.sub.3 = H, 40500
(.+-.6400).sup.a 61600 (.+-.7750).sup.d 1100 (.+-.210).sup.a 1900
(.+-.270).sup.b X = F) 7f (R.sub.1 = Et, R.sub.2 = H, R.sub.3 = H,
132000 (.+-.2200).sup.a 60500 (.+-.8160).sup.a 22600 12600 X = Cl)
(.+-.3400).sup.a (.+-.1800).sup.a 7g (R.sub.1 = .sup.iPr, R.sub.2 =
H, 6220 (.+-.790).sup.a 10300 (.+-.1300).sup.a 510 (.+-.75).sup.a
1230 (.+-.160).sup.b R.sub.3 = H, X = F) 7h (R.sub.1 = .sup.iPr,
R.sub.2 = H, 57600 (.+-.7560).sup.a 16900 (.+-.2200).sup.a 6700
(.+-.560).sup.a 2100 (.+-.310).sup.a R.sub.3 = H, X = Cl) 7i
(R.sub.1 = H, R.sub.2 = OMe, 91600 (.+-.12300).sup.a 17500
(.+-.2130).sup.a 8250 (.+-.980).sup.a 2180 (.+-.420).sup.a R.sub.3
= H, X = F) 7j (R.sub.1 = H, R.sub.2 = OMe, 130830 48140
(.+-.6300).sup.a 30120 4340 (.+-.620).sup.a R.sub.3 = H, X = Cl)
(.+-.35600).sup.a (.+-.3820).sup.a 7k (R.sub.1 = Me, R.sub.2 = OMe,
129100 210300 4430 (.+-.510).sup.a 14300 R.sub.3 = H, X = F)
(.+-.26300).sup.a (.+-.58200).sup.e (.+-.4800).sup.a 7l (R.sub.1 =
Me, R.sub.2 = OMe, 77900 (.+-.7500).sup.a 77800 19900 25300 R.sub.3
= H, X = Cl) (.+-.10900).sup.a (.+-.2120).sup.a (.+-.2410).sup.a 7m
(R.sub.1 = Me, R.sub.2 = OEt, 64400 (.+-.8320).sup.a 94450 1200
(.+-.150).sup.a 990 (.+-.110).sup.a R.sub.3 = H, X = F)
(.+-.17700).sup.f 7n (R.sub.1 = Me, R.sub.2 = OEt, 58900
(.+-.4500).sup.a 71850 (.+-.8320).sup.a 6470 (.+-.680).sup.a 2410
(.+-.320).sup.a R.sub.3 = H, X = Cl) 7o (R.sub.1 = Me, R.sub.2 = H,
124900 117300 1030 (.+-.90).sup.a 1230 (.+-.140).sup.a R.sub.3 =
OMe, X = F) (.+-.33500).sup.a (.+-.19500).sup.g 7p (R.sub.1 = Me,
R.sub.2 = H, 132800 54490 (.+-.6230).sup.a 13100 5410
(.+-.570).sup.a R3 = OMe, X = Cl) (.+-.27800).sup.a
(.+-.1450).sup.a 7q (R.sub.1 = Et, R.sub.2 = OMe, 59900
(.+-.6320).sup.a 50700 (.+-.4350).sup.a 1600 (.+-.180).sup.a 1580
(.+-.140).sup.a R.sub.3 = H, X = F) 7r (R.sub.1 = Et, R.sub.2 =
OMe, 107200 39440 (.+-.4200).sup.a 6180 (.+-.710).sup.a 2130
(.+-.210).sup.a R.sub.3 = H, X = Cl) (.+-.12300).sup.a 7s (R.sub.1
= H, R.sub.2 = H, 58410 (.+-.4320).sup.a 24880 (.+-.3120).sup.a
4570 (.+-.630).sup.a 15850 (.+-.1890) R.sub.3 = OMe, X = F) 7t
(R.sub.1 = H, R.sub.2 = H, 127700 60240 (.+-.8210).sup.a 17450
37800 R.sub.3 = OMe, X = Cl) (.+-.45300).sup.a (.+-.1560).sup.a
(.+-.4120).sup.a 7u (R.sub.1 = Et, R.sub.2 = H, 18930
(.+-.1900).sup.a 39460 (.+-.3870).sup.a 1980 (.+-.270).sup.a 790
(.+-.160).sup.a R.sub.3 = OMe, X = F) 7v (R.sub.1 = Et, R.sub.2 =
H, 60530 (.+-.8650).sup.a 46780 (.+-.7410).sup.a 12830 2930
(.+-.380).sup.a R.sub.3 = OMe, X = Cl) (.+-.1430).sup.a .sup.aA
single k.sub.obs was determined. .sup.bk.sub.inact/K.sub.I was
determined from a linear fit. .sup.ck.sub.inact = 2.24 min.sup.-1,
K.sub.I = 210 .mu.M. .sup.dk.sub.inact = 2.02 min.sup.-1, K.sub.I =
32.9 .mu.M. .sup.ek.sub.inact = 3.47 min.sup.-1, K.sub.I = 16.5
.mu.M. .sup.fk.sub.inact = 5.21 min.sup.-1, K.sub.I = 55.2 .mu.M.
.sup.gk.sub.inact = 2.42 min.sup.-1, K.sub.I = 20.7 .mu.M.
TABLE-US-00006 TABLE 3b Summary of isozyme selectivity for
compounds 7a-v Fold Fold Fold Fold PAD1 PAD2 PAD3 PAD4 Compound
Selectivity Selectivity Selectivity Selectivity 7a (R.sub.1 = H,
R.sub.2 = H, 24 3.3 1.0 10 R.sub.3 = H, X = F) 7b (R.sub.1 = H,
R.sub.2 = H, 6.5 2.3 1.0 2.1 R.sub.3 = H, X = Cl) 7c (R.sub.1 = Me,
R.sub.2 = H, 25 8.9 1.0 1.0 R.sub.3 = H, X = F) 7d (R.sub.1 = Me,
R.sub.2 = H, 61 3.1 2.0 1.0 R.sub.3 = H, X = Cl) 7e (R.sub.1 = Et,
R.sub.2 = H, 37 56 1.0 1.7 R.sub.3 = H, X = F) 7f (R.sub.1 = Et,
R.sub.2 = H, 10 4.8 1.8 1.0 R.sub.3 = H, X = Cl) 7g (R.sub.1 =
.sup.iPr, R.sub.2 = H, 12 20 1.0 2.4 R.sub.3 = H, X = F) 7h
(R.sub.1 = .sup.iPr, R.sub.2 = H, 27 8.0 3.2 1.0 R.sub.3 = H, X =
Cl) 7i (R.sub.1 = H, R.sub.2 = OMe, 42 8.0 3.8 1.0 R.sub.3 = H, X =
F) 7j (R.sub.1 = H, R.sub.2 = OMe, 30 11 6.9 1.0 R.sub.3 = H, X =
Cl) 7k (R.sub.1 = Me, R.sub.2 = 29 47 1.0 3.2 OMe, R.sub.3 = H, X =
F) 7l (R.sub.1 = Me, R.sub.2 = 3.9 3.9 1.0 1.3 OMe, R.sub.3 = H, X
= Cl) 7m (R.sub.1 = Me, R.sub.2 = 65 95 1.2 1.0 OEt, R.sub.3 = H, X
= F) 7n (R.sub.1 = Me, R.sub.2 = 24 30 2.7 1.0 OEt, R.sub.3 = H, X
= Cl) 7o (R.sub.1 = Me, R.sub.2 = H, 121 114 1.0 1.2 R.sub.3 = OMe,
X = F) 7p (R.sub.1 = Me, R.sub.2 = H, 25 10 2.4 1.0 R.sub.3 = OMe,
X = Cl) 7q (R.sub.1 = Et, R.sub.2 = 38 32 1.0 1.0 OMe, R.sub.3 = H,
X = F) 7r (R.sub.1 = Et, R.sub.2 = 50 19 2.9 1.0 OMe, R.sub.3 = H,
X = Cl) 7s (R.sub.1 = H, R.sub.2 = H, 13 5.4 1.0 3.5 R.sub.3 = OMe,
X = F) 7t (R.sub.1 = H, R.sub.2 = H, 7.3 3.5 1.0 2.2 R.sub.3 = OMe,
X = Cl) 7u (R.sub.1 = Et, R.sub.2 = H, 24 50 2.5 1.0 R.sub.3 = OMe,
X = F) 7v (R.sub.1 = Et, R.sub.2 = H, 21 16 4.4 1.0 R.sub.3 = OMe,
X = Cl)
##STR00016##
TABLE-US-00007 TABLE 4a k.sub.inact/K.sub.I values for compounds
8a-b' PAD1 k.sub.inact/K.sub.I PAD2 k.sub.inact/K.sub.I PAD3
k.sub.inact/K.sub.I PAD4 k.sub.inact/K.sub.I Compound
(M.sup.-1min.sup.-1) (M.sup.-1min.sup.-1) (M.sup.-1min.sup.-1)
(M.sup.-1min.sup.-1) 8a (R.sub.1 = H, R.sub.2 = H, R.sub.3 = H,
48400 (.+-.5210).sup.a 14400 (.+-.1320).sup.a 14900
(.+-.1250).sup.a 14900 R.sub.4 = Et, X = F) (.+-.1510).sup.a 8b
(R.sub.1 = H, R.sub.2 = H, R.sub.3 = H, 151700 53300
(.+-.6420).sup.a 35000 (.+-.2740).sup.a 48600 R.sub.4 = Et, X = Cl)
(.+-.28900).sup.a (.+-.4600).sup.a 8c (R.sub.1 = Me, R.sub.2 = H,
18500 (.+-.2230).sup.a 32760 (.+-.2890).sup.a 5750 (.+-.610).sup.a
3030 (.+-.280).sup.a R.sub.3 = H, R.sub.4 = Et, X = F) 8d (R.sub.1
= Me, R.sub.2 = H, 78910 (.+-.9480).sup.a 71350 (.+-.5210).sup.a
3770 (.+-.240).sup.a 4540 (.+-.330).sup.a R.sub.3 = H, R.sub.4 =
Et, X = Cl) 8e (R.sub.1 = Et, R.sub.2 = H, R.sub.3 = H, 24100
(.+-.1980).sup.a 40610 (.+-.4050).sup.a 1250 (.+-.130).sup.a 1240
(.+-.110).sup.a R.sub.4 = Et, X = F) 8f (R.sub.1 = Et, R.sub.2 = H,
R.sub.3 = H, 57100 (.+-.6050).sup.a 45100 (.+-.3030).sup.a 6870
(.+-.790).sup.a 3250 (.+-.280).sup.a R.sub.4 = Et, X = Cl) 8g
(R.sub.1 = .sup.iPr, R.sub.2 = H, 4700 (.+-.420).sup.a 10400
(.+-.890).sup.a 1600 (.+-.190).sup.a 470 (.+-.50).sup.a R.sub.3 =
H, R.sub.4 = Et, X = F) 8h (R.sub.1 = .sup.iPr, R.sub.2 = H, 26900
(.+-.1960).sup.a 16600 (.+-.1540).sup.a 4800 (.+-.520).sup.a 2430
(.+-.270).sup.a R.sub.3 = H, R.sub.4 = Et, X = Cl) 8i (R.sub.1 = H,
R.sub.2 = OMe, 80210 (.+-.9130).sup.a 23790 (.+-.2830).sup.a 9170
(.+-.1030).sup.a 750 (.+-.95).sup.a R.sub.3 = H, R.sub.4 = Et, X =
F) 8j (R.sub.1 = H, R.sub.2 = OMe, 91900 (.+-.5230).sup.a 43180
(.+-.5180).sup.a 18790 (.+-.2050).sup.a 18450 R.sub.3 = H, R.sub.4
= Et, X = Cl) (.+-.1750).sup.a 8k (R.sub.1 = Me, R.sub.2 = OMe,
70760 (.+-.18200).sup.a 365400 4320 (.+-.520).sup.a 4300
(.+-.410).sup.b R.sub.3 = H, R.sub.4 = Et, X = F) (.+-.84900).sup.c
8l (R.sub.1 = Me, R.sub.2 = OMe, 57800 (.+-.5550).sup.a 74660
(.+-.12330).sup.a 14840 (.+-.1620).sup.a 19400 R.sub.3 = H, R.sub.4
= Et, X = Cl) (.+-.2100).sup.a 8m (R.sub.1 = Me, R.sub.2 = OEt,
29800 (.+-.3140).sup.a 85600 (.+-.7950).sup.a 3470 (.+-.210).sup.a
3350 (.+-.150).sup.a R.sub.3 = H, R.sub.4 = Et, X = F) 8n (R.sub.1
= Me, R.sub.2 = OEt, 18100 (.+-.1930).sup.a 65170 (.+-.13200).sup.a
8280 (.+-.910).sup.a 1510 (.+-.80).sup.a R.sub.3 = H, R.sub.4 = Et,
X = Cl) 8o (R.sub.1 = Me, R.sub.2 = H, 50500 (.+-.6120).sup.a
133700 1070 (.+-.120).sup.a 4100 (.+-.330).sup.a R.sub.3 = OMe,
R.sub.4 = Et, X = F) (.+-.19800).sup.d 8p (R.sub.1 = Me, R.sub.2 =
H, 71100 (.+-.14200).sup.a 61500 (.+-.7800).sup.a 17750
(.+-.1630).sup.a 1900 (.+-.170).sup.a R.sub.3 = OMe, R.sub.4 = Et,
X = Cl) 8q (R.sub.1 = Me, R.sub.2 = OMe, 51470 (.+-.9100).sup.a
93300 (.+-.10760).sup.a 1600 (.+-.370).sup.a 4200 (.+-.470).sup.a
R.sub.3 = H, R.sub.4 = .sup.iPr, X = F) 8r (R.sub.1 = Me, R.sub.2 =
OMe, 43920 (.+-.3300).sup.a 51200 (.+-.8260).sup.a 14480
(.+-.3140).sup.a 12400 R.sub.3 = H, R.sub.4 = .sup.iPr, X = Cl)
(.+-.1520).sup.a 8s (R.sub.1 = Me, R.sub.2 = OMe, 45970
(.+-.7300).sup.a 94320 (.+-.14860).sup.a 1220 (.+-.280).sup.a 4500
(.+-.920).sup.a R.sub.3 = H, R.sub.4 = cyclopropyl, X = F) 8t
(R.sub.1 = Me, R.sub.2 = OMe, 40440 (.+-.1750).sup.a 57500
(.+-.7500).sup.a 9480 (.+-.1720).sup.a 8580 (.+-.3320).sup.a
R.sub.3 = H, R.sub.4 = cyclopropyl, X = Cl) 8u (R.sub.1 = Et,
R.sub.2 = OMe, 39100 (.+-.4210).sup.a 55980 (.+-.11400).sup.e 1400
(.+-.130).sup.a 2800 (.+-.190).sup.a R.sub.3 = H, R.sub.4 = Et, X =
F) 8v (R.sub.1 = Et, R.sub.2 = OMe, 54400 (.+-.4180).sup.a 46570
(.+-.3540).sup.a 5970 (.+-.460).sup.a 1750 (.+-.220).sup.a R.sub.3
= H, R.sub.4 = Et, X = Cl) 8w (R.sub.1 = H, R.sub.2 = H, 9360
(.+-.1020).sup.a 13540 (.+-.1890).sup.a 7630 (.+-.810).sup.a 2140
(.+-.190).sup.a R.sub.3 = OMe, R.sub.4 = Et, X = F) 8x (R.sub.1 =
H, R.sub.2 = H, 30270 (.+-.5660).sup.a 35400 (.+-.4280).sup.a 16000
(.+-.1890).sup.a 3890 (.+-.420).sup.a R.sub.3 = OMe, R.sub.4 = Et,
X = Cl) 8y (R.sub.1 = Et, R.sub.2 = H, 15170 (.+-.2130).sup.a 37780
(.+-.4250).sup.a 1480 (.+-.130).sup.a 540 (.+-.110).sup.a R.sub.3 =
OMe, R.sub.4 = Et, X = F) 8z (R.sub.1 = Et, R.sub.2 = H, 30070
(.+-.3190).sup.a 38210 (.+-.4310).sup.a 11850 (.+-.1540).sup.a 1410
(.+-.120).sup.a R.sub.3 = OMe, R.sub.4 = Et, X = Cl) 8a' (R.sub.1 =
Me, R.sub.2 = OMe, 59760 (.+-.6130).sup.a 212700 2010
(.+-.180).sup.a 2300 (.+-.410).sup.b R.sub.3 = H, R.sub.4 = Me, X =
F) (.+-.57650).sup.f 8b' (R.sub.1 = Me, R.sub.2 = OMe, 43150
(.+-.5100).sup.a 69880 (.+-.6430).sup.a 13650 (.+-.1410).sup.a 6340
(.+-.820).sup.a R.sub.3 = H, R.sub.4 = Me, X = F) .sup.aA single
k.sub.obs was determined. .sup.bk.sub.inact/K.sub.I was determined
from a linear fit. .sup.ck.sub.inact = 1.94 min.sup.-1, K.sub.I =
5.3 .mu.M. .sup.dk.sub.inact = 2.50 min.sup.-1, K.sub.I = 18.7
.mu.M. .sup.ek.sub.inact = 1.31 min.sup.-1, K.sub.I = 23.4 .mu.M.
.sup.fk.sub.inact = 2.73 min.sup.-1, K.sub.I = 12.8 .mu.M.
TABLE-US-00008 TABLE 4b Summary of isozyme selectivity for
compounds 8a-b' Fold Fold Fold Fold PAD1 PAD2 PAD3 PAD4 Compound
Selectivity Selectivity Selectivity Selectivity 8a (R.sub.1 = H,
R.sub.2 = H, 3.4 1.0 1.0 1.0 R.sub.3 = H, R.sub.4 = Et, X = F) 8b
(R.sub.1 = H, R.sub.2 = H, 4.3 1.5 1.0 1.4 R.sub.3 = H, R.sub.4 =
Et, X = Cl) 8c (R.sub.1 = Me, R.sub.2 = H, 6.1 11 1.9 1.0 R.sub.3 =
H, R.sub.4 = Et, X = F) 8d (R.sub.1 = Me, R.sub.2 = H, 21 19 1.0
1.2 R.sub.3 = H, R.sub.4 = Et, X = Cl) 8e (R.sub.1 = Et, R.sub.2 =
H, 19 33 1.0 1.0 R.sub.3 = H, R.sub.4 = Et, X = F) 8f (R.sub.1 =
Et, R.sub.2 = H, 18 14 2.1 1.0 R.sub.3 = H, R.sub.4 = Et, X = Cl)
8g (R.sub.1 = .sup.iPr, R.sub.2 = H, 10 22 3.4 1.0 R.sub.3 = H,
R.sub.4 = Et, X = F) 8h (R.sub.1 = .sup.iPr, R.sub.2 = H, 11 6.8
2.0 1.0 R.sub.3 = H, R.sub.4 = Et, X = Cl) 8i (R.sub.1 = H, R.sub.2
= OMe, 107 32 12 1.0 R.sub.3 = H, R.sub.4 = Et, X = F) 8j (R.sub.1
= H, R.sub.2 = OMe, 5.0 2.3 1.0 1.0 R.sub.3 = H, R.sub.4 = Et, X =
Cl) 8k (R.sub.1 = Me, R.sub.2 = OMe, 16 85 1.0 1.0 R.sub.3 = H,
R.sub.4 = Et, X = F) 8l (R.sub.1 = Me, R.sub.2 = OMe, 3.9 5.0 1.0
1.3 R.sub.3 = H, R.sub.4 = Et, X = Cl) 8m (R.sub.1 = Me, R.sub.2 =
OEt, 8.9 26 1.0 1.0 R.sub.3 = H, R.sub.4 = Et, X = F) 8n (R.sub.1 =
Me, R.sub.2 = OEt, 12 43 5.5 1.0 R.sub.3 = H, R.sub.4 = Et, X = Cl)
8o (R.sub.1 = Me, R.sub.2 = H, 47 125 1.0 3.8 R.sub.3 = OMe,
R.sub.4 = Et, X = F) 8p (R.sub.1 = Me, R.sub.2 = H, 37 32 9.3 1.0
R.sub.3 = OMe R.sub.4 = Et, X = Cl) 8q (R.sub.1 = Me, R.sub.2 =
OMe, 32 58 1.0 2.6 R.sub.3 = H, R.sub.4 = .sup.iPr, X = F) 8r
(R.sub.1 = Me, R.sub.2 = OMe, 3.5 4.1 1.2 1.0 R.sub.3 = H, R.sub.4
= .sup.iPr, X = Cl) 8s (R.sub.1 = Me, R.sub.2 = OMe, 38 77 1.0 3.8
R.sub.3 = H, R.sub.4 = cyclopropyl, X = F) 8t (R.sub.1 = Me,
R.sub.2 = OMe, 4.7 6.7 1.1 1.0 R.sub.3 = H, R.sub.4 = cyclopropyl,
X = Cl) 8u (R.sub.1 = Et, R.sub.2 = OMe, 28 40 2.0 1.0 R.sub.3 = H,
R.sub.4 = Et, X = F) 8v (R.sub.1 = Et, R.sub.2 = OMe, 31 27 3.4 1.0
R.sub.3 = H, R.sub.4 = Et, X = Cl) 8w (R.sub.1 = H, R.sub.2 = H,
4.4 6.3 3.6 1.0 R.sub.3 = OMe, R.sub.4 = Et, X = F) 8x (R.sub.1 =
H, R.sub.2 = H, 7.8 9.1 4.1 1.0 R.sub.3 = OMe, R.sub.4 = Et, X =
Cl) 8y (R.sub.1 = Et, R.sub.2 = H, 28 70 2.7 1.0 R.sub.3 = OMe,
R.sub.4 = Et, X = F) 8z (R.sub.1 = Et, R.sub.2 = H, 21 27 8.4 1.0
R.sub.3 = OMe, R.sub.4 = Et, X = Cl) 8a' (R.sub.1 = Me, R.sub.2 =
OMe, 30 106 1.0 1.1 R.sub.3 = H, R.sub.4 = Me, X = F) 8b' (R.sub.1
= Me, R.sub.2 = OMe, 6.8 11 2.2 1.0 R.sub.3 = H, R.sub.4 = Me, X =
F)
##STR00017##
TABLE-US-00009 TABLE 5a k.sub.inact/K.sub.I values for compounds
9a-f PAD1 k.sub.inact/K.sub.I PAD2 k.sub.inact/K.sub.I PAD3
k.sub.inact/K.sub.I PAD4 k.sub.inact/K.sub.I Compound
(M.sup.-1min.sup.-1) (M.sup.-1min.sup.-1) (M.sup.-1min.sup.-1)
(M.sup.-1min.sup.-1) 9a (R = Me, X = F) 2470 (.+-.270).sup.a 2400
(.+-.220).sup.b 1200 (.+-.90).sup.a 2430 (.+-.370).sup.b 9b (R =
Me, X = Cl) 19500 3310 (.+-.250).sup.b 5200 (.+-.710).sup.a 3850
(.+-.410).sup.b (.+-.2200).sup.a 9c (R = Et, X = F) 950
(.+-.70).sup.a 6100 (.+-.760).sup.b 3100 (.+-.440).sup.a 440
(.+-.90).sup.b 9d (R = Et, X = Cl) 12030 9150 (.+-.760).sup.b 4700
(.+-.920).sup.a 1300 (.+-.220).sup.b (.+-.1100).sup.a 9e (R =
.sup.iPr, X = F) 1420 (.+-.210).sup.a 3010 (.+-.290).sup.a 2100
(.+-.190).sup.a 160 (.+-.80).sup.a 9f (R = .sup.iPr, X = Cl) 3360
(.+-.520).sup.a 7150 (.+-.990).sup.a 4800 (.+-.520).sup.a 680
(.+-.110).sup.a .sup.aA single /k.sub.obs was determined.
.sup.bk.sub.inact/K.sub.I was determined from a linear fit.
TABLE-US-00010 TABLE 5b Summary of isozyme selectivity for
compounds 9a-f Fold Fold Fold Fold PAD1 PAD2 PAD3 PAD4 Compound
Selectivity Selectivity Selectivity Selectivity 9a (R = Me, X = F)
2.1 2.0 1.0 2.0 9b (R = Me, X = Cl) 5.9 1.0 1.6 1.2 9c (R = Et, X =
F) 2.2 14 7.0 1.0 9d (R = Et, X = Cl) 9.3 7.0 3.6 1.0 9e (R =
.sup.iPr, X = F) 8.9 19 13 1.0 9f (R = .sup.iPr, X = Cl) 4.9 11 7.1
1.0
##STR00018##
TABLE-US-00011 TABLE 6a k.sub.inact/K.sub.I values for compounds
10a-h and 11a-h PAD1 k.sub.inact/K.sub.I PAD2 k.sub.inact/K.sub.I
PAD3 k.sub.inact/K.sub.I PAD4 k.sub.inact/K.sub.I Compound
(M.sup.-1min.sup.-1) (M.sup.-1min.sup.-1) (M.sup.-1min.sup.-1)
(M.sup.-1min.sup.-1) 10a + 11a (R = H, X = F) 12600
(.+-.1450).sup.a 10400 (.+-.930).sup.b 20030 27300 (.+-.1980).sup.a
(.+-.2910).sup.b 10b + 11b (R = H, X = Cl) 57300 (.+-.9800).sup.a
34300 32800 41400 (.+-.4200).sup.b (.+-.2890).sup.a
(.+-.3800).sup.b 10c + 11c (R = Me, X = F) 28850 (.+-.2760).sup.a
27700 29800 3250 (.+-.670).sup.a (.+-.3100).sup.a (.+-.1980).sup.a
10d + 11d (R = Me, 65800 (.+-.9500).sup.a 63800 44100 14400 X = Cl)
(.+-.9800).sup.a (.+-.8200).sup.a (.+-.4200).sup.a 10e + 11e (R =
Et, X = F) 29900 (.+-.3400).sup.a 43300 9500 (.+-.1020).sup.a 1930
(.+-.180).sup.a (.+-.3500).sup.a 10f + 11f (R = Et, X = Cl) 71600
(.+-.8700).sup.a 78300 30100 32090 (.+-.6400).sup.a
(.+-.2870).sup.a (.+-.3050).sup.a 10g + 11g (R = .sup.iPr, X = F)
10050 (.+-.1100).sup.a 13600 10300 (.+-.960).sup.a 380
(.+-.50).sup.b (.+-.1200).sup.b 10h + 11h (R = .sup.iPr, 57300
(.+-.4070).sup.a 26900 7100 (.+-.830).sup.a 3700 (.+-.320).sup.b X
= Cl) (.-+.2900).sup.b .sup.aA single k.sub.obs was determined.
.sup.bk.sub.inact/K.sub.I was determined from a linear fit.
TABLE-US-00012 TABLE 6b Summary of isozyme selectivity for
compounds 10a-h and 11a-h Fold Fold Fold Fold PAD1 PAD2 PAD3 PAD4
Compound Selectivity Selectivity Selectivity Selectivity 10a + 11a
(R = H, 1.2 1.0 1.9 2.6 X = F) 10b + 11b (R = H, 1.7 1.0 1.0 1.3 X
= Cl) 10c + 11c (R = Me, 8.9 8.5 9.2 1.0 X = F) 10d + 11d (R = Me,
4.6 4.4 3.1 1.0 X = Cl) 10e + 11e (R = Et, 15 22 4.9 1.0 X = F) 10f
+ 11f (R = Et, 2.4 2.6 1.0 1.1 X = Cl) 10g + 11g (R = .sup.iPr, 26
36 27 1.0 X = F) 10h + 11h (R = .sup.iPr, 15 7.3 1.9 1.0 X =
Cl)
[0213] FIG. 1 shows exemplary concentration dependent labeling of
recombinant PAD2 with BB--F-Yne (5u) (FIG. 1A) and with BB--Cl-Yne
(5v) (FIG. 1C) and the limit of detection (LOD) of BB--F-Yne (5u)
and of BB--Cl-Yne (5v) for PAD2, (FIG. 1B) and (FIG. 1D),
respectively. Decreasing concentrations of PAD2 treated with
BB--F-Yne (5u) and "Clicked" with TAMRA-N.sub.3. Decreasing
concentrations of PAD2 were treated with BB--Cl-Yne (5v) and
"Clicked" with TAMRA-N.sub.3.
[0214] FIG. 2 shows exemplary in vitro labeling of the four active
PAD isozymes with BB--F-Yne (5u). FIG. 2A shows concentration
dependent labeling of recombinant PAD1 with BB--F-Yne (5u). PAD1
was treated with increasing concentrations of BB--F-Yne (5u) and
then "Clicked" with TAMRA-N.sub.3. FIG. 2B shows concentration
Concentration dependent labeling of recombinant PAD2 with BB--F-Yne
(5u). PAD2 was treated with increasing concentrations of BB--F-Yne
(5u) and then "Clicked" with TAMRA-N.sub.3. FIG. 2C shows
concentration dependent labeling of recombinant PAD3 with BB--F-Yne
(5u). PAD3 was treated with increasing concentrations of BB--F-Yne
(5u) and then "Clicked" with TAMRA-N.sub.3. FIG. 2D shows
concentration dependent labeling of recombinant PAD4 with BB--F-Yne
(5u). PAD4 was treated with increasing concentrations of BB--F-Yne
(5u) and then "Clicked" with TAMRA-N.sub.3.
[0215] FIG. 3 shows exemplary cellular labeling of PAD2 with
BB--F-Yne (5u) in ionophore stimulated HEK293T/PAD2 cells. FIG. 3A
shows HEK293T/PAD2 cells treated with increasing concentrations of
BB--F-Yne (5u) for 1 h. The cells were then harvested and probe
labeled proteins were tagged with TAMRA-N.sub.3 to facilitate
visualization after SDS-PAGE. (B&C) HEK293T/PAD2 cells were
treated with increasing concentrations of BB--F-Yne (5u) for 1 h.
The cells were then harvested and probe labeled proteins were
tagged with Biotin-TEV-N.sub.3. Biotin tagged proteins were then
isolated on streptavidin agarose and the eluted proteins were
probed for PAD2 (FIG. 3B) or biotinylated proteins using
streptavidin-RP (FIG. 3C).
[0216] FIG. 4 shows exemplary cellular labeling of PAD2 with
BB--Cl-Yne (5v) in ionophore stimulated HEK293T/PAD2 cells. FIG. 4A
shows HEK293T/PAD2 cells treated with increasing concentrations of
BB--Cl-Yne (5v) for 1 h. The cells were then harvested and probe
labeled proteins were tagged with TAMRA-N.sub.3 to facilitate
visualization after SDS-PAGE. FIG. 4B-4C show HEK293T/PAD2 cells
treated with increasing concentrations of BB--Cl-Yne (5v) for 1 h.
The cells were then harvested and probe labeled proteins were
tagged with Biotin-TEV-N.sub.3. Biotin tagged proteins were then
isolated on streptavidin agarose and the eluted proteins were
probed for PAD2 (FIG. 4B) or biotinylated proteins using
streptavidin-RP (FIG. 4C).
[0217] FIG. 5 shows exemplary cellular labeling of PAD4 with
BB--F-Yne (5u). FIG. 5A shows HEK293T/PAD4 cells were treated with
increasing concentrations of BB--F-Yne (5u) for 1 h. The cells were
then harvested and probe labeled proteins were tagged with
TAMRA-N.sub.3 to facilitate visualization after SDS-PAGE. FIG. 5B
shows HEK293T/PAD4 cells treated with increasing concentrations of
BB--F-Yne (5u) for 1 h. The cells were then harvested and probe
labeled proteins were tagged with Biotin-TEV-N.sub.3. Biotin tagged
proteins were then isolated on streptavidin agarose and the eluted
proteins were probed for PAD2.
EXPERIMENTAL
Chemistry
[0218] .sup.1H NMR were recorded at 400 (Bruker DRX-400 with a
H/C/P/F QNP gradient probe) or 500 MHz (Bruker BioSpin 500 MHz
Advance III Digital NMR) spectrometer and .sup.13C NMR spectra were
recorded at 100 or 125 MHz; chemical shifts are reported in .delta.
(ppm) relative to the internal chloroform-d (CDCl.sub.3, 7.26 ppm)
or methanol-d (CD.sub.3OD, 3.31 ppm). ESI (HRMS) were recorded with
a Micromass Q-TQF I. The purity of all compounds was determined to
be >95% purity as determined by .sup.1H NMR and .sup.13C NMR
spectra, unless otherwise noted. TLC was performed on glass backed
silica gel plates (Uniplate) with spots visualized by UV light. All
solvents were reagent grade and, when necessary, were purified and
dried by standard methods. Concentrations of solutions after
reactions and extractions involved the use of a rotary evaporator
operating at reduced pressure.
Chemicals and Proteins
[0219] Dithiothreitol (DTT),
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),
ammonium iron (III) sulfate dodecahydrate,
tris(2-carboxyethyl)phosphine (TCEP), and thiosemicarbazide were
acquired from Sigma-Aldrich. Diacetylmonooxime (DAMO),
N-.alpha.-benzoyl-L-arginine ethyl ester (BAEE), and
N-.alpha.-benzoyl-L-arginine amide (BAA) were obtained from Acros.
Detailed synthetic procedures are described below. PADs 1, 2, 3,
and 4 were purified as reported. (Causey, et al. 2011 J Med Chem
54, 6919-6935; Knuckley, et al. 2010 Biochemistry 49, 4852-4863.)
HEK293T and HEK293T cells stably expressing human PAD2
(HEK293T/PAD2) or PAD4 (HEK293T/PAD4) were cultured as previously
described. (Lewallen, et al. 2014 ACS Chem Biol 9, 913-921.)
Biotin-TEV-N.sub.3 was synthesized as previously reported.
(Weerapana, et al., 2007 Nat Protoc 2, 1414-1425.) TAMRA-N.sub.3
was obtained from Lumiprobe.
Inactivation Kinetics
[0220] Inactivation kinetic parameters were determined by
incubating PAD1, 2, or 4 (2.0 .mu.M) or PAD3 (5.0 .mu.M) in a
pre-warmed (10 min; 37.degree. C.) inactivation mixture (50 mM
HEPES, 10 mM CaCl.sub.2, and 2 mM DTT, pH 7.6, with a final volume
of 60 .mu.L) containing various concentrations of inhibitor.
Aliquots were removed at various time points and added to a
pre-warmed (10 min, 37.degree. C.) reaction mixture (50 mM HEPES,
50 mM NaCl, 10 mM CaCl.sub.2, 2 mM DTT, and 10 mM BAEE or 10 mM BAA
in the case of PAD3; pH 7.6). After 15 min, reactions were quenched
in liquid nitrogen and citrulline production quantified using the
COLDER assay. (Kearney, et al. 2005 Biochemistry 44, 10570-10582;
Knipp, et al. 2000 Anal Biochem 286, 257-264.) Data were plotted as
a function of time and fit to eq 1,
v=v.sub.oe.sup.-kt eq 1,
using GraFit version 5.0.11, where v is velocity, v.sub.o is
initial velocity, k (or k.sub.obs) is the pseudo-first order rate
constant of inactivation, and t is time. When saturation was
reached upon plotting k.sub.obs versus inactivator concentration,
the data were fit to eq 2,
k.sub.obs=k.sub.inact[I]/(K.sub.I+[I]) eq 2,
using GraFit version 5.0.11, where k.sub.inact corresponds to the
maximal rate of inactivation and K.sub.I is the concentration of
inhibitor that gives half-maximal inactivation. If the plot of
k.sub.obs versus [I] was linear and did not saturate, then the
value for k.sub.inact/K.sub.I equaled the slope of the line. In
Vitro Labeling of PADs with "Clickable" Probes BB--F-Yne (5u),
BB--Cl--Yne (5v) and BIFYne (5w)
[0221] Increasing concentrations of 5u-w (0 to 10 .mu.M) were
incubated with recombinant PADs (1 .mu.M) in the presence of
CaCl.sub.2 in 1.times.PBS (2 mM) at 37.degree. C. for 1 h. The
probe labeled enzymes were coupled to TAMRA-N.sub.3 (20 .mu.M) in
the presence of 1.times.TBTA (0.31 mM), sodium ascorbate (2 mM) and
freshly prepared CuSO.sub.4 (1 mM). The tubes were gently tumbled
for 1 h. The reactions were quenched with 6.times.SDS loading
buffer and separated by SDS-PAGE (12.5% gel). The bands were
visualized by scanning the gel in a typhoon scanner (approximate
excitation/emission maxima .about.546/579, respectively).
In Vitro Labeling of PAD2 with BB--F-Yne (5u) and BB--Cl-Yne (5v)
to Determine their Limit of Detection (LOD)
[0222] BB--F-Yne (5u) or BB--Cl-Yne (5v) (10 .mu.M) were incubated
with decreasing concentrations of recombinant PAD2 (1.0 to 0.025
.mu.M) in the presence of CaCl.sub.2 in 1.times.PBS (2 mM) at
37.degree. C. for 1 h. The probe labeled enzymes were coupled to
TAMRA-N.sub.3 (20 .mu.M) in the presence of 1.times.TBTA (0.31 mM),
sodium ascorbate (2 mM) and freshly prepared CuSO.sub.4 (1 mM). The
tubes were gently tumbled for 1 h. The reactions were quenched with
6.times.SDS loading buffer and separated by SDS-PAGE (12.5% gel).
The bands were visualized by scanning the gel in a typhoon scanner
(approximate excitation/emission maxima .about.546/579,
respectively).
Cellular Labeling of PAD2 with BB--F-Yne (5u) and BB--Cl-Yne (5v)
in Ionomycin Stimulated HEK293T/PAD2 Cells
[0223] HEK293T cells stably expressing human PAD2 (HEK293T PAD2)
were cultured. Cells were grown to .about.80% confluence
(8.times.10.sup.6 cells), trypsinized, and quenched trypsin
activity with complete media. The cells were harvested by
centrifugation at 1000.times.g for 2 min and washed 4.times. with
1.times.PBS. Cells were resuspended in PBS at 8.times.10.sup.6
cells/mL and 4.times.10.sup.5 cells were added to 0.65 mL tubes for
subsequent assays.
[0224] HEK293T PAD2 cells were treated with increasing
concentrations of BB--F-Yne (5u) or BB--Cl-Yne (5v) (0 to 50 .mu.M)
in the presence of CaCl.sub.2 (2 mM) at 37.degree. C. After 20 min,
ionomycin (10 .mu.M) was added and the cells incubated for 1 h
before addition of Triton X-100 (1% final in PBS) and sonication at
4.degree. C. for 1 h. Lysates were cleared by centrifugation at
21,000.times.g for 15 min. The soluble protein fraction was
isolated and quantified by the DC assay (Biorad). Lysates (2
.mu.g/.mu.L, 50 .mu.L total) were "Clicked" with TAMRA-N.sub.3 (20
.mu.M), 1.times.TBTA (0.31 mM), sodium ascorbate (2 mM) and freshly
prepared CuSO.sub.4 (1 mM). The tubes were gently tumbled for 2 h.
The reactions were quenched by the addition of 6.times.SDS loading
buffer and separated by SDS-PAGE (12.5% gel). The bands were
visualized by scanning the gel in a typhoon scanner (approximate
excitation/emission maxima .about.546/579, respectively).
[0225] Lysates (2 .mu.g/.mu.L, 500 .mu.L total) were also "Clicked"
with Bio-TEV-N.sub.3 (100 .mu.M), 1.times.TBTA (0.31 mM), sodium
ascorbate (2 mM) and freshly prepared CuSO.sub.4 (1 mM). The tubes
were gently tumbled for 2 h. The cloudy solution was transferred to
a microconcentrator (10 kDa molecular weight cutoff) and
centrifuged at 10000.times.g for 5 min at 4.degree. C. to remove
the excess biotin azide. The protein was then resolubilized in
1.times.PBS with 0.2% SDS. Resolubilized protein samples were
incubated with 100 .mu.L streptavidin-agarose beads (Thermo
Scientific) at 4.degree. C. for 16 h. The solutions were then
incubated at rt for 1 h. The beads were washed with 0.2% SDS/PBS,
PBS, and water. The beads were pelleted by centrifugation
(1,600.times.g, 3 min) between washes. To the washed beads,
2.times.SDS loading buffer was added and heated to 95.degree. C.
heat block for 15 min. The resolubilized protein was separated by
SDS-PAGE (12.5% gel) and transferred to PVDF membranes (Biorad) at
80 V for 60 min. The membranes were analyzed for PAD2 and for
biotinylated proteins.
1. General Procedure for Synthesis of Benzene-1,2-Diamine
Intermediates
##STR00019##
[0227] To a stirred solution of 1a-c (1.0 eq) in DMF was added
NHR.sub.3 (3.0 eq) in a sealed tube. The reaction mixture was then
heated to 70.degree. C. for 12 h, cooled, and diluted with water.
The product was then filtered, washed with water, dried under
vacuum, and obtained in 78-89% yield. This product was then
dissolved in a THF/EtOH/H.sub.2O (3:1:1) solvent system, and cooled
to 0.degree. C. A solution of Na.sub.2S.sub.2O.sub.4 in water was
then added dropwise to the stirred mixture. The reaction mixture
was then warmed to rt and allowed to stir for 4 h. Upon completion,
saturated NaHCO.sub.3 solution was added followed by EtOAc
extraction. The combined organic layers were then washed with
deionized water. The organic layer was then separated, dried over
anhydrous Na.sub.2SO.sub.4, filtered, and concentrated to give
desired products 2a-d in 64-76% yield.
N.sup.1-ethyl-3-methoxybenzene-1,2-diamine (2a)
##STR00020##
[0229] .sup.1H NMR (CDCl.sub.3; 400 MHz): .delta. 6.78 (t, J=8.2
Hz, 1H), 6.41 (t, J=8.4 Hz, 2H), 3.85 (s, 3H), 3.19-3.14 (m, 2H),
1.30 (t, J=7.3 Hz, 3H). .sup.13C NMR (CDCl.sub.3; 100 MHz): .delta.
148.3, 138.6, 122.6, 119.6, 117.1, 111.8, 106.0, 105.3, 101.7,
55.8, 55.6, 43.6, 38.9, 15.1. LRMS m/z calculated for
C.sub.9H.sub.14N.sub.2O (M+H.sup.+) 167.1; found 167.2.
3-ethoxy-N.sup.1-methylbenzene-1,2-diamine (2b)
##STR00021##
[0231] .sup.1H NMR (CDCl.sub.3; 400 MHz): .delta. 6.78 (t, J=8.3
Hz, 1H), 6.43-6.37 (m, 2H), 4.09-4.03 (m, 2), 2.87 (s, 3H), 1.42
(t, J=7.4 Hz, 3H). .sup.13C NMR (CDCl.sub.3; 100 MHz): .delta.
147.4, 139.6, 122.9, 119.6, 104.4, 102.9, 64.1, 31.2, 15.1. LRMS
m/z calculated for C.sub.9H.sub.14N.sub.2O (M+H.sup.+) 167.1; found
167.2.
4-methoxy-N.sup.1-methylbenzene-1,2-diamine (2c)
##STR00022##
[0233] .sup.1H NMR (CDCl.sub.3; 400 MHz): .delta. 6.89 (dd, J=2.2
Hz, J=8.7 Hz, 1H), 6.52 (d, J=8.2 Hz, 1H), 6.27-6.24 (m, 1H), 3.79
(s, 3H), 3.66 (s, 3H). .sup.13C NMR (CDCl.sub.3; 100 MHz): .delta.
153.6, 143.6, 136.4, 132.1, 118.2, 113.2, 112.8, 109.6, 103.8,
103.2, 102.2, 55.5, 31.8, 31.0, 29.3. LRMS m/z calculated for
C.sub.8H.sub.12N.sub.2O (M+H.sup.+) 152.1; found 152.2.
4-methoxy-N.sup.1-ethylbenzene-1,2-diamine (2d)
##STR00023##
[0235] .sup.1H NMR (CDCl.sub.3; 400 MHz): .delta. 7.10 (dd, J=2.3
Hz, J=8.2 Hz, 1H), 6.54 (d, J=8.2 Hz, 1H), 6.30-6.27 (m, 1H), 3.77
(s, 3H), 3.00 (m, 2H), 1.19 (t, 3H). .sup.13C NMR (CDCl.sub.3; 100
MHz): .delta. 153.8, 137.1, 131.0, 118.3, 114.2, 111.5, 109.1,
104.1, 103.8, 103.1, 102.8, 101.7, 55.9, 55.5, 39.7, 38.5, 15.2,
14.9. LRMS m/z calculated for C.sub.9H.sub.14N.sub.2O (M+H.sup.+)
167.1; found 167.1.
2. General Procedure for Synthesis of Benzimidazole
Intermediates
##STR00024##
[0237] To a stirred solution of Fmoc-Orn(Boc)-OH (3, 1.0 eq) in DMF
was added N.sup.1R.sub.1benzene-1,2-diamine (1.0 eq), DIPEA (3.0
eq), followed by HBTU (2.0 eq) and HOBt, (2.0 eq). This reaction
mixture was stirred at rt under N.sub.2 atmosphere for 12 h, and
then diluted with water. The product was filtered, washed with
water, and dried under vacuum, and obtained in 81-92% yield. This
product was dissolved in glacial AcOH/toluene (1:1) and heated at
80.degree. C. for 16 h. The reaction mixture was evaporated to
dryness and diluted with water to precipitate the desired product
(Fmoc-Orn(Boc)-benzimidazole) which was isolated by vacuum
filtration in 84-96% yield. This compound was then treated with 20%
piperidine in DMF for 20 min at rt to cleave the Fmoc group and the
reaction was diluted with hexanes to remove the byproduct. The oily
product was further purified by column chromatography using MeOH
and CH.sub.2Cl.sub.2 as the eluent to give 4a-j in 45-56%
yield.
tert-butyl-(4-amino-4-(1H-benzo[d]imidazol-2-yl)butyl)carbamate-L-ornithin-
e (4a)
##STR00025##
[0239] .sup.1H NMR (CDCl.sub.3; 400 MHz): .delta. 7.55-7.52 (m,
2H), 7.22-7.19 (m, 2H), 4.33 (t, J=6.7 Hz, 1H), 3.31-3.22 (m, 1H),
3.11-3.02 (m, 1H), 2.01-1.92 (m, 1H), 1.83-1.75 (m, 1H), 1.69-1.49
(m, 2H), 1.44 (s, 9H). .sup.13C NMR (CDCl.sub.3; 100 MHz): .delta.
156.5, 122.1, 115.1, 79.5, 49.6, 39.5, 34.4, 28.3, 26.5. LRMS m/z
calculated for C.sub.16H.sub.24N.sub.4O.sub.2 (M+H.sup.+) 305.2;
found 305.1.
tert-butyl-(4-amino-4-(1-methyl-benzo[d]imidazol-2-yl)butyl)carbamate-L-or-
nithine (4b)
##STR00026##
[0241] .sup.1H NMR (CDCl.sub.3; 400 MHz): .delta. 7.73 (dd, J=2.6
Hz, 6.7 Hz, 1H), 7.33-7.29 (m, 1H), 7.28-7.25 (m, 2H), 4.28 (t,
J=6.8 Hz, 1H), 3.79 (s, 3H), 3.18-3.13 (m, 2H), 2.10-2.01 (m, 1H),
1.94-1.83 (m, 1H), 1.71-1.53 (m, 2H), 1.41 (s, 9H). .sup.13C NMR
(CDCl.sub.3; 100 MHz): .delta. 149.6, 145.2, 139.2, 127.0, 126.1,
122.6, 113.6, 100.0, 51.5, 43.8, 37.7, 33.7, 32.1, 29.8. LRMS m/z
calculated for C.sub.17H.sub.26N.sub.4O.sub.2 (M+H.sup.+) 319.2;
found 319.2.
tert-butyl-(4-amino-4-(1-ethyl-benzo[d]imidazol-2-yl)butyl)carbamate-L-orn-
ithine (4c)
##STR00027##
[0243] .sup.1H NMR (CDCl.sub.3; 400 MHz): .delta. 7.67 (dd, J=2.2
Hz, J=7.2 Hz, 1H), 7.32-7.30 (m, 1H), 7.24-7.19 (m, 2H), 4.27-4.22
(m, 1H), 4.21-4.12 (m, 2H), 3.10-3.07 (m, 2H), 2.05-1.96 (m, 1H),
1.91-1.82 (m, 1H), 1.64-1.46 (m, 2H), 1.37 (m, 12H). .sup.13C NMR
(CDCl.sub.3; 100 MHz): .delta. 156.2, 155.9, 141.8, 134.3, 122.7,
122.0, 119.3, 109.5, 79.0, 48.1, 39.9, 38.5, 34.2, 28.3, 26.3,
15.1. LRMS m/z calculated for C.sub.18H.sub.28N.sub.4O.sub.2
(M+H.sup.+) 333.2; found 333.3.
tert-butyl-(4-amino-4-(1-isopropyl-benzo[d]imidazol-2-yl)butyl)carbamate-L-
-ornithine (4d)
##STR00028##
[0245] .sup.1H NMR (CDCl.sub.3; 400 MHz): .delta. 7.71 (dd, J=2.4
Hz, 7.3 Hz, 1H), 7.52 (dd, J=2.1 Hz, J=6.3 Hz, 1H), 7.21-7.19 (m,
2H), 4.89-4.77 (m, 1H), 4.19 (t, J=6.8 Hz, 1H), 3.18-3.08 (m, 2H),
2.06-1.96 (m, 1H), 1.86-1.77 (m, 1H), 1.65-1.62 (m, 6H), 1.60-1.52
(m, 2H), 1.40 (s, 9H). .sup.13C NMR (CDCl.sub.3; 100 MHz): .delta.
156.7, 155.9, 142.8, 133.7, 121.9, 121.6, 119.5, 111.9, 78.8, 49.0,
47.5, 40.3, 34.4, 28.2, 26.4, 21.3. LRMS m/z calculated for
C.sub.19H.sub.30N.sub.4O.sub.2 (M+H.sup.+) 347.2; found 347.2.
tert-butyl-(4-amino-4-(5,6-difluoro-benzo[d]imidazol-2-yl)butyl)carbamate--
L-ornithine (4e)
##STR00029##
[0247] .sup.1H NMR (CDCl.sub.3; 400 MHz): .delta. 7.28 (t, J=8.6
Hz, 2H), 4.38-4.35 (m, 1H), 3.31-3.22 (m, 1H), 3.11-3.03 (m, 1H),
1.99-1.90 (m, 1H), 1.85-1.77 (m, 1H), 1.68-1.58 (m, 1H), 1.56-1.49
(m, 1H), 1.43 (s, 9H). .sup.13C NMR (CDCl.sub.3; 100 MHz): .delta.
156.8, 148.9, 147.0, 133.4, 102.5, 79.8, 49.5, 39.4, 33.7, 28.4,
26.5. LRMS m/z calculated for
C.sub.16H.sub.22F.sub.2N.sub.4O.sub.2(M+H.sup.+) 341.2; found
341.1.
tert-butyl-(4-amino-4-(4-methoxy-benzo[d]imidazol-2-yl)butyl)carbamate-L-o-
rnithine (4f)
##STR00030##
[0249] .sup.1H NMR (CDCl.sub.3; 400 MHz): .delta. 7.08 (s, 2H),
6.58 (s, 1H), 4.58-4.53 (m, 1H), 3.87 (s, 3H), 3.14-3.06 (m, 1H),
3.03-2.96 (m, 1H), 2.11-2.00 (m, 1H), 1.97-1.90 (m, 1H), 1.51-1.39
(m, 2H), 1.36 (s, 9H). .sup.13C NMR (CDCl.sub.3; 100 MHz): .delta.
177.1, 162.6, 156.5, 149.4, 138.2, 129.6, 123.6, 106.9, 102.8,
79.4, 55.8, 55.5, 49.6, 39.5, 36.5, 32.6, 31.4, 28.3, 26.3, 22.5.
LRMS m/z calculated for C.sub.17H.sub.26N.sub.4O.sub.3 (M+H.sup.+)
335.2; found 335.2.
tert-butyl-(4-amino-4-(4-methoxy-1-methyl-benzo[d]imidazol-2-yl)butyl)carb-
amate-L-ornithine (4g)
##STR00031##
[0251] .sup.1H NMR (CDCl.sub.3; 400 MHz): .delta. 7.19 (t, J=8.5
Hz, 1H), 6.92 (d, J=8.6 Hz, 1H), 6.67 (d, J=8.3 Hz, 1H), 4.23-4.19
(m, 1H), 4.00 (s, 3H), 3.75 (s, 3H), 3.17-3.11 (m, 2H), 2.05-1.96
(m, 1H), 1.92-1.82 (m, 1H), 1.70-1.53 (m, 2H), 1.40 (s, 9H).
.sup.13C NMR (CDCl.sub.3; 100 MHz): .delta. 156.2, 151.3, 137.3,
132.0, 123.1, 102.5, 102.3, 79.2, 55.6, 48.5, 40.0, 34.1, 30.0,
28.2, 26.5. LRMS m/z calculated for C.sub.18H.sub.28N.sub.4O.sub.3
(M+H.sup.+) 349.2; found 349.1.
tert-butyl-(4-amino-4-(4-methoxy-1-ethyl-benzo[d]imidazol-2-yl)butyl)carba-
mate-L-ornithine (4h)
##STR00032##
[0253] .sup.1H NMR (CDCl.sub.3; 400 MHz): .delta. 7.15 (t, J=8.4
Hz, 1H), 6.92 (d, J=7.5 Hz, 1H), 6.62 (d, J=8.4 Hz, 1H), 4.23-4.12
(m, 1H), 3.91 (s, 3H), 3.11-3.03 (m, 2H), 2.01-1.92 (m, 1H),
1.88-1.79 (m, 1H), 1.61-1.44 (m, 2H), 1.38 (t, J=7.5 Hz, 3H), 1.35
(s, 9H). .sup.13C NMR (CDCl.sub.3; 100 MHz): .delta. 156.1, 155.2,
151.23, 135.9, 132.1, 123.2, 102.8, 79.0, 55.5, 50.5, 48.2, 39.9,
38.4, 34.4, 28.2, 26.3, 15.1. LRMS m/z calculated for
C.sub.19H.sub.30N.sub.4O.sub.3 (M+H.sup.+) 363.2; found 363.3.
tert-butyl-(4-amino-4-(5-methoxy-1H-benzo[d]imidazol-2-yl)butyl)carbamate--
L-ornithine (4i)
##STR00033##
[0255] .sup.1H NMR (CDCl.sub.3; 400 MHz): .delta. 7.32 (d, J=8.4
Hz, 1H), 6.91 (s, 1H), 6.75 (dd, J=2.3 Hz, J=8.6 Hz, 1H), 4.19 (m,
1H), 3.71 (s, 3H), 3.39 (s, 3H), 1.91-1.83 (m, 1H), 1.76-1.69 (m,
1H), 1.48-1.42 (m, 2H), 1.34 (s, 9H). .sup.13C NMR (CDCl.sub.3; 100
MHz): .delta. 156.7, 156.2, 138.2, 133.2, 115.8, 111.7, 97.7, 79.2,
55.8, 50.3, 49.8, 39.7, 33.8, 28.5, 26.2. LRMS m/z calculated for
C.sub.17H.sub.26N.sub.4O.sub.3 (M+H.sup.+) 335.2; found 335.3.
tert-butyl-(4-amino-4-(5-methoxy-1-methyl-benzo[d]imidazol-2-yl)butyl)carb-
amate-L-ornithine (4j)
##STR00034##
[0257] .sup.1H NMR (CDCl.sub.3; 400 MHz): .delta. 7.21 (d, J=2.4
Hz, 1H), 7.17 (d, J=8.7 Hz, 1H), 6.90 (dd, J=2.4 Hz, J=8.7 Hz, 1H),
4.20-4.18 (m, 1H), 3.82 (s, 3H), 3.72 (s, 3H), 3.15-3.10 (m, 2H),
2.05-1.95 (m, 1H), 1.90-1.80 (m, 1H), 1.68-1.49 (m, 2H), 1.40 (s,
9H). .sup.13C NMR (CDCl.sub.3; 100 MHz): .delta. 157.1, 156.3,
155.9, 142.5, 130.4, 112.4, 109.7, 101.5, 79.0, 55.6, 48.3, 39.9,
33.9, 29.9, 28.5, 26.6. LRMS m/z calculated for
C.sub.18H.sub.28N.sub.4O.sub.3 (M+H.sup.+) 349.2; found 349.2.
tert-butyl-(4-amino-4-(5-methoxy-1-ethyl-benzo[d]imidazol-2-yl)butyl)carba-
mate-L-ornithine (4k)
##STR00035##
[0259] .sup.1H NMR (CDCl.sub.3; 400 MHz): .delta. 7.15-7.12 (m,
2H), 6.83 (dd, J=2.4 Hz, J=8.6 Hz, 1H), 4.17-4.10 (m, 2H),
4.08-4.05 (m, 1H), 3.76 (s, 3H), 2.00-1.92 (m, 1H), 1.84-1.77 (m,
1H), 1.61-1.53 (m, 1H), 1.50-1.43 (m, 1H), 1.34 (s, 12H). .sup.13C
NMR (CDCl.sub.3; 100 MHz): .delta. 156.1, 142.9, 129.2, 112.5,
109.9, 101.8, 55.9, 50.3, 48.2, 40.1, 38.5, 34.5, 28.3, 26.5, 15.4.
LRMS m/z calculated for C.sub.19H.sub.30N.sub.4O.sub.3 (M+H.sup.+)
363.2; found 363.2.
tert-butyl-(4-amino-4-(4-ethoxy-1-methyl-benzo[d]imidazol-2-yl)butyl)carba-
mate-L-ornithine (4l)
##STR00036##
[0261] .sup.1H NMR (CDCl.sub.3; 400 MHz): .delta. 7.16 (t, J=7.8
Hz, 1H), 6.89 (d, J=7.7 Hz, 1H), 6.67 (d, J=7.8 Hz, 1H), 4.31-4.26
(m, 1H), 4.25-4.22 (m, 2H), 3.75 (s, 3H), 3.16-3.11 (m, 2H),
2.04-1.95 (m, 1H), 1.90-1.82 (m, 1H), 1.69-1.55 (m, 2H), 1.52 (t,
J=7.2 Hz, 3H), 1.40 (s, 9H). .sup.13C NMR (CDCl.sub.3; 100 MHz):
.delta. 156.0, 150.2, 137.5, 132.4, 123.1, 103.7, 101.7, 79.1,
64.1, 48.7, 39.8, 33.7, 30.2, 28.4, 26.5, 14.6. LRMS m/z calculated
for C.sub.19H.sub.30N.sub.4O.sub.3 (M+H.sup.+) 363.2; found
363.1.
3a. General Procedure for Synthesis of Benzimidazole
haloacetamidines 5a-b..sup.a
##STR00037##
[0263] To a stirred solution of 4a (1.0 eq) in THF/H.sub.2O (1:1)
was added TEA (3.0 eq) followed by benzoyl chloride (1.0 eq) and
allowed to stir at rt for 3 h. Solvents were evaporated and the
crude product was purified by reverse phase HPLC using
MeCN:H.sub.2O (0.5% TFA) as the eluent to give the product in
72-76% yield. This product was then treated with TFA to remove the
Boc group giving the Bz-Orn-benzimidazole intermediate. The solvent
was then evaporated to dryness and the crude material was dried in
vacuo. To a stirred solution of the corresponding
Bz-Orn-benzimidazole intermediate in dry MeOH was added TEA (4.0
eq) followed by ethyl haloacetimidate HCl (2.0 eq). The reaction
was stirred under N.sub.2 at rt for 3 h. Solvents were then
evaporated under reduced pressure and the crude product was
purified by reverse phase HPLC using MeCN:H.sub.2O (0.5% TFA) as an
eluent to give compound 5a-b in 72-78% yield.
(N1-benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1H-benzo[d]imidazol-2-yl)-L-orni-
thine) (5a)
##STR00038##
[0265] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.00-7.97 (m,
2H), 7.81-7.76 (m, 5H), 5.66-5.63 (m, 1H), 5.30 (d, J=45.6 Hz, 2H),
3.55-3.50 (m, 2H), 2.39-2.34 (m, 2H), 2.05-.192 (m, 1H), 1.90-1.83
(m, 1H). .sup.13C NMR (100 MHz, CD.sub.3 OD) .delta. 170.5, 155.4,
134.1, 133.5, 133.3, 129.7, 128.8, 127.2, 115.2, 79.8, 78.1, 42.8,
30.6, 25.2. HRMS m/z calculated for C.sub.20H.sub.23FN.sub.5O
(M+H.sup.+) 368.1878; found 368.1880.
(N1-benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1H-benzo[d]imidazol-2-yl)-L-orni-
thine) (5b)
##STR00039##
[0267] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.06-7.97 (m,
2H), 7.85-7.78 (m, 2H), 7.62-7.59 (m, 3H), 7.58-7.50 (m, 2H),
5.67-5.63 (m, 1H), 4.39 (s, 2H), 3.54-3.41 (m, 2H), 2.40-2.35 (m,
2H), 2.05-1.92 (m, 1H), 1.90-1.81 (m, 1H). .sup.13C NMR (100 MHz,
CD.sub.3 OD) .delta. 170.5, 164.5, 162.5, 155.3, 134.1, 133.6,
133.0, 129.7, 128.8, 127.4, 115.1, 43.3, 40.1, 30.4, 25.0. HRMS m/z
calculated for C.sub.20H.sub.23ClN.sub.5O (M+H.sup.+) 384.1583;
found 384.1588.
3b. General Procedure for Synthesis of Benzimidazole
haloacetamidines 5c-da
##STR00040##
[0269] To a stirred solution of 4a (1.0 eq) in THF/H.sub.2O (1:1)
was added TEA (3.0 eq) followed by naphthoyl chloride (1.0 eq) and
allowed to stir at rt for 3 h. Solvents were evaporated and the
crude product was purified by reverse phase HPLC using
MeCN:H.sub.2O (0.5% TFA) as the eluent to give the product in
68-73% yield. This product was then treated with TFA to remove the
Boc group giving the Naphthyl-Om-benzimidazole intermediate. The
solvent was then evaporated to dryness and the crude material was
dried in vacuo. To a stirred solution of the corresponding
Naphthyl-Orn-benzimidazole intermediate in dry MeOH was added TEA
(4.0 eq) followed by ethyl haloacetimidate HCl (2.0 eq). The
reaction was stirred under N.sub.2 at rt for 3 h. Solvents were
then evaporated under reduced pressure and the crude product was
purified by reverse phase HPLC using MeCN:H.sub.2O (0.5% TFA) as an
eluent to give compound 5c-d in 30-40% yield.
(N1-[2-naphthoyl-N5-(2-fluoro-1-iminoethyl)-1-(1H-benzo[d]imidazol-2-yl)-L-
-ornithine) (5c)
##STR00041##
[0271] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.58 (s, 1H),
8.11-7.93 (m, 4H), 7.81-7.74 (m, 2H), 7.66-7.56 (m, 4H), 5.74-5.70
(m, 1H), 5.30 (d, J=45.6 Hz, 2H), 3.58-3.50 (m, 2H), 2.44-2.40 (m,
2H), 2.10-1.86 (m, 2H). .sup.13C NMR (100 MHz, CD.sub.3 OD) .delta.
170.5, 164.6, 164.4, 136.6, 133.9, 132.9, 131.3, 130.2, 129.6,
129.5, 129.3, 128.8, 128.1, 127.4, 125.0, 115.1, 79.8, 78.1, 42.8,
30.5, 25.2. HRMS m/z calculated for C.sub.24H.sub.25FN.sub.5O
(M+H.sup.+) 418.2035; found 418.2040.
(N1-[2-naphthoyl-N5-(2-chloro-1-iminoethyl)-1-(1H-benzo[d]imidazol-2-yl)-L-
-ornithine) (5d)
##STR00042##
[0273] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.58 (s, 1H),
8.12-7.92 (m, 4H), 7.81-7.73 (m, 2H), 7.67-7.55 (m, 4H), 5.74-5.70
(m, 1H), 4.42 (s, 2H), 3.57-3.49 (m, 2H), 2.43-2.39 (m, 2H),
2.10-1.85 (m, 2H). .sup.13C NMR (100 MHz, CD.sub.3 OD) .delta.
170.4, 169.4, 164.9, 163.0, 162.6, 154.6, 139.1, 133.8, 133.7,
131.7, 131.2, 130.3, 128.9, 127.2, 115.2, 43.3, 40.1, 30.4, 25.0.
HRMS m/z calculated for C.sub.24H.sub.25ClN.sub.5O (M+H.sup.+)
434.1739; found 434.1741.
3c. General Procedure for Synthesis of Benzimidazole
haloacetamidines 5e-w..sup.a
##STR00043##
[0275] To a stirred solution of 4a-e, g (1.0 eq) in
CH.sub.2Cl.sub.2 was added TEA (3.0 eq) followed by
biphenyl-4-carbonyl chloride (1.0 eq) and allowed to stir at rt for
3 h. Solvents were evaporated and the crude product was purified by
reverse phase HPLC using MeCN:H.sub.2O (0.5% TFA) as the eluent to
give the product in 69-80% yield. This product was then treated
with TFA to remove the Boc group giving the
4-Ph-Bz-Orn-benzimidazole intermediate. The solvent was then
evaporated to dryness and the crude material was dried in vacuo. To
a stirred solution of the corresponding 4-Ph-Bz-Orn-benzimidazole
intermediate in dry MeOH was added TEA (4.0 eq) followed by ethyl
haloacetimidate HCl (2.0 eq). The reaction was stirred under
N.sub.2 at rt for 3 h. Solvents were then evaporated under reduced
pressure and the crude product was purified by reverse phase HPLC
using MeCN:H.sub.2O (0.5% TFA) as an eluent to give compound 5a-p
in 53-75% yield.
(N1-[1,4'-Phenyl]benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1H-benzo[d]imidazol-
-2-yl)-L-ornithine) 5e)
##STR00044##
[0277] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.07 (d, J=8.4
Hz, 2H), 7.87-7.76 (m, 4H), 7.77 (d, J=8.4 Hz, 2H), 7.69-7.58 (m,
2H), 7.51-7.47 (m, 2H), 7.43-7.39 (m, 1H), 5.70-5.56 (m, 1H), 5.30
(d, J=45.2 Hz, 2H), 3.51-3.49 (m, 2H), 2.42-2.36 (m, 2H), 2.06-1.83
(m, 2H). .sup.13C NMR (100 MHz, CD.sub.3 OD) .delta. 170.2, 164.5,
155.4, 146.5, 141.0, 132.8, 132.7, 130.1, 129.5, 129.3, 128.2,
127.5, 115.3, 79.8, 78.1, 42.8, 30.5, 25.2. HRMS m/z calculated for
C.sub.26H.sub.27FN.sub.5O (M+H.sup.+) 444.2191; found 444.2194.
(N1-[1,4'-Phenyl]benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1H-benzo[d]imidazol-
-2-yl)-L-omithine)
##STR00045##
[0279] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.06 (d, J=8.4
Hz, 2H), 7.79 (dd, J=3.2 Hz, J=8.4 Hz, 2H), 7.74 (d, J=8.4 Hz, 2H),
7.67-7.64 (m, 2H), 7.58 (dd, J=3.2 Hz, J=8.4 Hz, 2H), 7.48-7.44 (m,
2H), 7.39-7.36 (m, 1H), 5.71-5.57 (m, 1H), 4.40 (s, 2H), 3.56-3.43
(m, 2H), 2.47-2.33 (m, 2H), 2.09-2.01 (m, 1H), 1.98-1.83 (m, 1H).
.sup.13C NMR (100 MHz, CD.sub.3 OD) .delta. 170.3, 164.8, 155.4,
146.5, 141.0, 132.6, 132.5, 130.1, 129.5, 129.3, 128.2, 127.6,
115.1, 43.3, 40.1 (2C), 30.6, 25.1. HRMS m/z calculated for
C.sub.26H.sub.27ClN.sub.5O (M+H.sup.+) 460.1896; found
460.1892.
(N1-[1,4'-Phenyl]benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-methyl-benzo[d]im-
idazol-2-yl)-L-ornithine) (52)
##STR00046##
[0281] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.01 (d, J=8.4
Hz, 2H), 7.87 (dd, J=2.3 Hz, J=8.1 Hz, 1H), 7.81 (dd, J=2.3 Hz,
J=7.2 Hz, 1H), 7.75 (d, J=8.3 Hz, 2H), 7.66 (d, J=8.3 Hz, 2H),
7.63-7.58 (m, 2H), 7.47 (t, J=7.3 Hz, 2H), 7.39 (t, J=7.8 Hz, 1H),
5.78-5.74 (m, 1H), 5.27 (d, J=45.4 Hz, 2H), 4.17 (s, 3H), 3.54-3.44
(m, 2H), 2.45-2.26 (m, 2H), 2.06-2.01 (m, 1H), 1.91-1.80 (m, 1H).
.sup.13C NMR (100 MHz, CD.sub.3 OD) .delta. 168.5, 163.2, 162.9,
161.4, 160.9, 153.0, 145.1, 139.6, 133.6, 131.21, 128.7 (2C),
127.9, 127.9, 126.8, 126.7, 125.5, 125.4, 115.2, 111.6, 78.5, 76.7,
45.7, 41.4, 30.3, 28.9, 23.7. HRMS m/z calculated for
C.sub.27H.sub.28FN.sub.5O (M+H.sup.+) 458.2351; found 458.2349.
(N1-[1,4'-Phenyl]benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-methyl-benzo[d]im-
idazol-2-yl)-L-ornithine) (5h)
##STR00047##
[0283] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.00 (d, J=8.3
Hz, 2H), 7.86 (dd, J=2.4 Hz, J=7.6 Hz, 1H), 7.80 (dd, J=2.4 Hz,
J=7.2 Hz, 1H), 7.75 (d, J=8.7 Hz, 2H), 7.68-7.65 (m, 2H), 7.62-7.57
(m, 2H), 7.47 (t, J=7.2 Hz, 2H), 7.39 (t, J=7.2 Hz, 1H), 5.77-5.73
(m, 1H), 4.38 (s, 2H), 4.14 (s, 3H), 3.53-3.41 (m, 2H), 2.46-2.29
(m, 2H), 2.08-1.97 (m, 1H), 1.93-1.81 (m, 1H). .sup.13C NMR (100
MHz, CD.sub.3 OD) .delta. 168.5, 163.4, 161.3, 155.3, 152.9, 145.1,
139.5, 134.9, 133.9, 133.7, 131.3, 128.7 (2C), 127.9, 127.9, 126.8,
126.7, 125.3, 125.3, 115.3, 111.5, 45.7, 42.0, 38.7, 30.2, 29.0,
23.5. HRMS m/z calculated for C.sub.27H.sub.28ClN.sub.5O
(M+H.sup.+) 474.2055; found 474.2056.
(N1-[1,4'-Phenyl]benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-ethyl-benzo[d]imi-
dazol-2-yl)-L-omithine) (5i)
##STR00048##
[0285] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 7.99 (d, J=8.6
Hz, 2H), 7.88 (dd, J=2.1 Hz, J=7.2 Hz, 1H), 7.81 (dd, J=2.2 Hz,
J=7.1 Hz, 1H), 7.75 (d, J=8.6 Hz, 2H), 7.66 (d, J=7.7 Hz, 2H),
7.61-7.56 (m, 2H), 7.47 (t, J=7.3 Hz, 2H), 7.39 (t, J=7.7 Hz, 1H),
5.78-5.74 (m, 1H), 5.27 (d, J=45.2 Hz, 2H), 4.66-4.58 (m, 2H),
3.54-3.45 (m, 2H), 2.45-2.36 (m, 1H), 2.34-2.25 (m, 1H), 2.06-1.97
(m, 1H), 1.93-1.81 (m, 1H), 1.41 (t, J=7.1 Hz, 3H). .sup.13C NMR
(100 MHz, CD.sub.3 OD) .delta. 168.2, 163.1, 163.0, 152.5, 145.1,
139.5, 132.8, 132.3, 131.9, 131.4, 128.7 (2C), 127.9, 126.8, 126.7,
125.1, 124.9, 123.2, 115.9, 111.6, 78.5, 76.7, 45.5, 41.6, 39.7,
29.4, 23.8, 13.6. HRMS m/z calculated for C.sub.28H.sub.30FN.sub.5O
(M+H) 472.2507; found 472.2506.
(N1-[1,4'-Phenyl]benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-ethyl-benzo[d]imi-
dazol-2-yl)-L-ornithine) (5j)
##STR00049##
[0287] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 7.99 (d, J=8.3
Hz, 2H), 7.90 (dd, J=2.1 Hz, J=7.4 Hz, 1H), 7.81 (dd, J=2.4 Hz,
J=7.3 Hz, 1H), 7.75 (d, J=8.3 Hz, 2H), 7.66 (d, J=8.4 Hz, 2H),
7.62-7.56 (m, 2H), 7.46 (t, J=7.2 Hz, 2H), 7.40 (t, J=7.3 Hz, 1H),
5.78-5.74 (m, 1H), 4.67-4.60, 4.37 (s, 2H), 3.52-3.42 (m, 2H),
2.46-2.36 (m, 1H), 2.34-2.26 (m, 1H), 2.08-1.97 (m, 1H), 1.93-1.82
(m, 1H), 1.54 (t, J=7.5 Hz, 3H). .sup.13C NMR (100 MHz, CD.sub.3
OD) .delta. 168.3, 163.4, 161.3, 161.0, 152.4, 145.1, 139.6, 134.6,
132.7, 131.3, 128.7 (2C), 127.9, 126.8, 126.7, 125.2, 125.1, 115.7,
111.7, 100.0, 56.1, 45.5, 42.1, 39.8, 38.7, 29.3, 23.7, 13.5. HRMS
m/z calculated for C.sub.28H.sub.30ClN.sub.5O (M+H.sup.+) 488.2212;
found 488.2214.
(N1-[1,4'-Phenyl]benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-isopropyl-benzo[d-
]imidazol-2-yl)-L-ornithine) (5k)
##STR00050##
[0289] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.06-8.02 (m,
1H), 7.98 (d, J=8.5 Hz, 2H), 7.83-7.79 (m, 1H), 7.75 (d, J=8.5 Hz,
2H), 7.66 (dd, J=2.1 Hz, J=8.1 Hz, 2H), 7.57-7.53 (m, 2H), 7.47 (t,
J=7.4 Hz, 2H), 7.39 (t, J=7.4 Hz, 1H), 5.80-5.76 (m, 1H), 5.28 (d,
J=45.3 Hz, 2H), 5.21-5.16 (m, 1H), 3.57-3.44 (m, 2H), 2.45-2.36 (m,
1H), 2.35-2.27 (m, 1H), 2.06-1.96 (m, 1H), 1.93-1.84 (m, 1H), 1.81
(d, J=6.6 Hz, 3H), 1.71 (d, J=6.6 Hz, 3H). .sup.13C NMR (100 MHz,
CD.sub.3 OD) .delta. 168.4, 166.0, 165.1, 162.5, 161.4, 152.1,
145.1, 139.5, 133.0, 131.1, 130.6, 128.7 (2C), 127.9, 126.8, 126.6,
125.7, 125.4, 115.1, 114.4, 78.4, 76.7, 50.8, 46.0, 41.4, 29.0,
23.7, 19.6 (2C). HRMS m/z calculated for C.sub.29H.sub.32FN.sub.5O
(M+H.sup.+) 486.2664; found 486.2666.
(N1-[1,4'-Phenyl]benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-isopropyl-benzo[d-
]imidazol-2-yl)-L-ornithine) (5l)
##STR00051##
[0291] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.05-8.03 (m,
1H), 7.99 (d, J=8.3 Hz, 2H), 7.83-7.79 (m, 1H), 7.75 (d, J=8.4 Hz,
2H), 7.67 (dd, J=2.2 Hz, J=8.3 Hz, 2H), 7.58-7.53 (m, 2H), 7.47 (t,
J=7.3 Hz, 2H), 7.39 (t, J=7.6 Hz, 1H), 5.80-5.76 (m, 1H), 5.25-5.15
(m, 1H), 4.38 (s, 2H), 3.55-3.42 (m, 2H), 2.46-2.36 (m, 1H),
2.35-2.27 (m, 1H), 2.07-1.96 (m, 1H), 1.93-1.85 (m, 1H), 1.81 (d,
J=6.7 Hz, 3H), 1.72 (d, J=6.5 Hz, 3H). .sup.13C NMR (100 MHz,
CD.sub.3 OD) .delta. 168.4, 166.2, 165.3, 163.3, 160.9, 152.1,
145.1, 139.5, 131.0, 130.4, 128.6 (2C), 127.9, 126.8, 126.7, 125.9,
125.5, 115.6, 115.0, 114.5, 52.8, 50.9, 46.1, 41.9, 38.6, 29.0,
23.6, 19.6 (2C). HRMS m/z calculated for C.sub.29H.sub.32ClN.sub.5O
(M+H.sup.+) 502.2368; found 502.2370.
(N1-[1,4'-Phenyl]benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(4-methoxy-1-methyl--
benzo[d]imidazol-2-yl)-L-omithine) (5m)
##STR00052##
[0293] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 7.97 (d, J=8.5
Hz, 2H), 7.75 (d, J=8.6 Hz, 2H), 7.66 (dd, J=1.8 Hz, J=7.2 Hz, 2H),
7.49-7.37 (m, 4H), 7.28 (d, J=8.4 Hz, 2H), 6.99 (d, J=8.1 Hz, 2H),
5.69-5.64 (m, 1H), 5.27 (d, J=45.1 Hz, 2H), 4.03 (s, 3H), 4.01 (s,
3H), 3.51-3.41 (m, 2H), 2.34-2.28 (m, 2H), 2.00-1.89 (m, 1H),
1.86-1.75 (m, 1H). .sup.13C NMR (100 MHz, CD.sub.3 OD) .delta.
168.5, 163.0, 162.9, 161.3, 160.9, 152.1, 148.1, 144.9, 139.5,
134.6, 131.0, 128.6, 127.9, 126.8, 126.7, 122.7, 117.9, 115.0,
105.9, 103.6, 78.3, 76.6, 55.1, 46.0, 41.4, 30.7, 28.7, 23.5. HRMS
m/z calculated for C.sub.28H.sub.30FN.sub.5O.sub.2(M+H.sup.+)
488.2456; found 488.2455.
(N1-[1,4'-Phenyl]benzoyl-N5-(2-chloro-1-iminoethyl)-1-(4-methoxy-1-methyl--
benzo[d]imidazol-2-yl)-L-ornithine) (5n)
##STR00053##
[0295] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 7.98 (d, J=8.5
Hz, 2H), 7.74 (d, J=8.6 Hz, 2H), 7.65 (dd, J=1.8 Hz, J=8.4 Hz, 2H),
7.48-7.36 (m, 4H), 7.29 (d, J=8.4 Hz, 2H), 7.00 (d, J=8.2 Hz, 2H),
5.69-5.64 (m, 1H), 4.36 (s, 2H), 4.03 (s, 3H), 4.02 (s, 3H),
3.49-3.39 (m, 2H), 2.35-2.28 (m, 2H), 2.00-1.91 (m, 1H), 1.85-1.75
(m, 1H). .sup.13C NMR (100 MHz, CD.sub.3 OD) .delta. 168.5, 163.2,
158.1, 157.7, 152.1, 148.5, 145.0, 139.6, 134.8, 131.1, 128.6,
127.9, 127.9, 126.8, 126.7, 126.6, 105.7, 103.6, 85.2, 55.1, 46.0,
43.0, 42.0, 38.6, 30.5, 28.8, 28.6, 23.5. HRMS m/z calculated for
C.sub.28H.sub.30ClN.sub.5O.sub.2(M+H.sup.+) 504.2161; found
504.2162.
(N1-4'-[1,1'-Phenyl]benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1H-benzo[d]imida-
zol-2-yl)-L-ornithine) 5o)
##STR00054##
[0297] .sup.1H NMR (CD.sub.3 OD; 500 MHz): .delta. 7.94 (d, J=9.3
Hz, 2H), 7.67-7.63 (m, 4H), 7.47-7.43 (m, 2H), 7.12-7.09 (m, 2H),
5.56-5.53 (m, 1H), 5.16 (d, J=45.1 Hz, 2H), 3.44-3.35 (m, 2H),
2.29-2.24 (m, 2H), 1.94-1.86 (m, 1H), 1.81-1.72 (m, 1H). .sup.13C
NMR (125 MHz, CD.sub.3 OD) .delta. 168.7, 163.9, 163.1, 163.0,
162.0, 161.7, 161.3, 153.9, 143.8, 136.0, 131.9, 131.4, 128.7,
128.6, 128.1, 126.5, 125.7, 115.4, 115.3, 113.8, 109.2, 78.2, 76.9,
41.4, 29.1, 23.7. HRMS m/z calculated for
C.sub.26H.sub.25F.sub.2N.sub.5O (M+H.sup.+) 462.2100; found
462.2097.
(N1-4'-F-[1,1'-Phenyl]benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1H-benzo[d]imi-
dazol-2-yl)-L-ornithine) (5p)
##STR00055##
[0299] .sup.1H NMR (CD.sub.3 OD; 500 MHz): .delta. 7.94 (d, J=9.3
Hz, 2H), 7.68-7.63 (m, 4H), 7.61-7.57 (m, 2H), 7.47-7.44 (m, 2H),
7.12-7.08 (m, 2H), 5.57-5.54 (m, 1H), 4.28 (s, 2H), 3.42-3.32 (m,
2H), 2.30-2.24 (m, 2H), 1.94-1.86 (m, 1H), 1.80-1.72 (m, 1H).
.sup.13C NMR (125 MHz, CD.sub.3 OD) .delta. 168.6, 164.0, 163.3,
162.0, 162.0, 161.8, 161.4, 153.9, 143.9, 136.0, 135.9, 131.9,
131.3, 128.8, 128.7, 128.1, 126.5, 125.8, 117.8, 115.5, 115.3,
113.8, 41.9, 38.7, 29.1, 23.6. HRMS m/z calculated for
C.sub.26H.sub.25FClN.sub.5O (M+H.sup.+) 478.1804; found
478.1803.
(N1-[1,1'-Phenyl]benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(5,6-difluoro-1H-ben-
zo[d]imidazol-2-yl)-L-ornithine) (5q)
##STR00056##
[0301] .sup.1H NMR (CD.sub.3 OD; 500 MHz): .delta. 7.92 (d, J=9.4
Hz, 2H), 7.63 (d, J=9.4 Hz, 2H), 7.56-7.50 (m, 4H), 7.38-7.34 (m,
2H), 7.29-7.26 (m, 1H), 5.50-5.47 (m, 1H), 5.17 (d, J=45.3 Hz, 2H),
3.43-3.34 (m, 2H), 2.27-2.14 (m, 2H), 1.91-1.82 (m, 1H), 1.79-1.69
(m, 1H). .sup.13C NMR (125 MHz, CD.sub.3 OD) .delta. 168.6, 163.1,
162.9, 161.4, 161.3, 160.8, 156.1, 150.0, 149.9, 148.1, 147.8,
144.9, 139.6, 131.6, 129.9, 128.7, 128.0, 127.8, 126.7, 102.3,
102.2, 78.3, 76.8, 41.5, 29.5, 23.7. HRMS m/z calculated for
C.sub.26H.sub.24F.sub.3N.sub.5O (M+H.sup.+) 480.2006; found
480.1998.
(N1-[1,1'-Phenyl]benzoyl-N5-(2-chloro-1-iminoethyl)-1-(5,6-difluoro-1H-ben-
zo[d]imidazol-2-yl)-L-ornithine) (5r)
##STR00057##
[0303] .sup.1H NMR (CD.sub.3 OD; 500 MHz): .delta. 7.92 (d, J=9.3
Hz, 2H), 7.63 (d, J=9.4 Hz, 2H), 7.56-7.49 (m, 4H), 7.37-7.34 (m,
2H), 7.29-7.26 (m, 1H), 5.50-5.47 (m, 1H), 4.27 (s, 2H), 3.38-3.33
(m, 2H), 2.28-2.15 (m, 2H), 1.90-1.82 (m, 1H), 1.79-1.71 (m, 1H).
.sup.13C NMR (125 MHz, CD.sub.3 OD) .delta. 168.6, 163.3, 161.3,
161.0, 156.2, 150.0, 149.8, 148.1, 147.9, 144.9, 139.6, 131.5,
129.9, 128.71, 128.0, 127.8, 126.7, 126.7, 102.4, 102.4, 102.3,
102.2, 42.0, 38.7, 29.5, 23.6. HRMS m/z calculated for
C.sub.26H.sub.24F.sub.2ClN.sub.5O (M+H.sup.+) 496.1710; found
496.1706.
(N1-4'-F-[1,1'-Phenyl]benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(5,6-difluoro-1-
H-benzo[d]imidazol-2-yl)-L-omithine) (5s)
##STR00058##
[0305] .sup.1H NMR (CD.sub.3 OD; 500 MHz): .delta. 7.93-7.90 (d,
J=9.1 Hz, 2H), 7.63-7.60 (d, J=9.1 Hz, 2H), 7.59-7.55 (m, 2H),
7.52-7.49 (m, 2H), 7.11-7.07 (m, 2H), 5.49-5.46 (m, 1H), 5.16 (d,
J=45.4 Hz, 2H), 3.42-3.33 (m, 2H), 2.28-2.14 (m, 2H), 1.92-1.81 (m,
1H), 1.79-1.70 (m, 1H). .sup.13C NMR (125 MHz, CD.sub.3 OD) .delta.
168.6, 163.9, 163.1, 162.8, 162.1, 161.2, 161.0, 156.1, 150.0,
149.8, 148.0, 147.8, 143.8, 136.0, 131.6, 130.0, 128.7, 128.6,
128.0, 126.5, 115.5, 115.3, 102.4, 102.3, 102.3, 102.2, 78.3, 76.9,
41.4, 29.5, 23.8. HRMS m/z calculated for
C.sub.26H.sub.23F.sub.4N.sub.5O (M+H.sup.+) 498.1911; found
498.1903.
(N1-4'-F-[1,1'-Phenyl]benzoyl-N5-(2-chloro-1-iminoethyl)-1-(5,6-difluoro-1-
H-benzo[d]imidazol-2-yl)-L-omithine) (5t)
##STR00059##
[0307] .sup.1H NMR (CD.sub.3 OD; 500 MHz): .delta. 7.91 (d, J=9.4
Hz, 2H), 7.61 (d, J=9.3 Hz, 2H), 7.59-7.56 (m, 2H), 7.52-7.50 (m,
2H), 7.11-7.07 (m, 2H), 5.50-5.46 (m, 1H), 4.27 (s, 2H), 3.39-3.31
(m, 2H), 2.28-2.14 (m, 2H), 1.92-1.82 (m, 1H), 1.79-1.70 (m, 1H).
.sup.13C NMR (125 MHz, CD.sub.3 OD) .delta. 168.6, 163.9, 163.3,
162.0, 161.2, 160.9, 156.2, 150.0, 149.8, 148.0, 147.8, 143.8,
135.9, 131.6, 129.8, 128.7, 128.0, 126.6, 115.4, 115.3, 102.4,
102.3, 102.3, 102.2, 42.0, 38.7, 29.6, 23.6. HRMS m/z calculated
for C.sub.26H.sub.23F.sub.3ClN.sub.5O (M+H.sup.+) 514.1616; found
514.1612.
(N1-4'-ethynyl-[1,1'-Phenyl]benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1H-benzo-
[d]imidazol-2-yl)-L-ornithine) (5u)
##STR00060##
[0309] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.08 (d, J=8.4
Hz, 2H), 7.81-7.69 (m, 4H), 7.65-7.63 (m, 2H), 7.58-7.57 (m, 4H),
5.67 (t, J=7.6 Hz, 1H), 5.30 (d, J=45.2 Hz, 2H), 3.61 (s, 1H),
3.56-3.48 (m, 2H), 2.41-2.35 (m, 2H), 2.07-1.85 (m, 2H). .sup.13C
NMR (100 MHz, CD.sub.3 OD) .delta. 170.0, 164.5, 164.4, 163.2,
162.8, 155.4, 145.3, 141.2, 133.7, 133.4, 133.2, 129.9, 129.6,
128.2, 128.1, 127.2, 123.7, 115.2, 84.0, 79.9, 79.8, 78.1, 71.4,
55.8, 43.8, 42.8, 30.6, 25.1. HRMS m/z calculated for
C.sub.28H.sub.26FN.sub.5O (M+H.sup.+) 468.2192; found 468.2191.
(N1-4'-ethynyl-[1,1'-Phenyl]benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1H-benzo-
[d]imidazol-2-yl)-L-ornithine) (5v)
##STR00061##
[0311] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.07 (d, J=8.4
Hz, 2H), 7.82-7.78 (m, 4H), 7.72-7.69 (m, 2H), 7.60-7.57 (m, 4H),
5.67 (t, J=7.6 Hz, 1H), 4.39 (s, 2H), 3.61 (s, 1H), 3.52-3.47 (m,
2H), 2.42-2.36 (m, 2H), 2.07-1.83 (m, 2H). .sup.13C NMR (100 MHz,
CD.sub.3 OD) .delta. 170.0, 164.8, 155.3, 145.3, 141.2, 133.7,
133.3, 133.2, 129.6, 128.2, 128.1, 127.3, 123.7, 115.2, 84.0, 79.9,
43.4, 40.1, 30.5, 25.0. HRMS m/z calculated for
C.sub.28H.sub.26ClN.sub.5O (M+H.sup.+) 484.1896; found
484.1902.
(N1-4-ethynyl-benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-isopropyl-benzo[d]im-
idazol-2-yl)-L-ornithine) (5w)
##STR00062##
[0313] .sup.1H NMR (CD.sub.3 OD; 500 MHz): .delta. 8.13-8.11 (m,
1H), 7.93 (d, J=8.3 Hz, 2H), 7.87-7.85 (m, 1H), 7.65-7.62 (m, 2H),
7.58 (d, J=8.3 Hz, 2H), 5.80-5.77 (m, 1H), 5.35-5.29 (m, 1H), 5.30
(d, J=45.5 Hz, 2H), 3.74 (s, 1H), 3.58-3.48 (m, 2H), 2.50-2.42 (m,
1H), 2.34-2.26 (m, 1H), 2.11-2.02 (m, 1H), 1.94-1.87 (m, 1H), 1.85
(d, J=6.8 Hz, 3H), 1.79 (d, J=7.2 Hz, 3H). .sup.13C NMR (125 MHz,
CD.sub.3 OD) .delta. 167.9, 163.1, 163.0, 161.1, 152.1, 132.4,
131.8, 131.5, 130.2, 127.5, 126.6, 126.3, 125.9, 114.8, 114.6,
81.9, 80.3, 78.3, 76.9, 51.4, 46.3, 41.4, 28.9, 23.8, 19.6. HRMS
m/z calculated for C.sub.25H.sub.28FN.sub.5O (M+H.sup.+) 434.2349;
found 434.2348.
4. General Procedure for Synthesis of Benzimidazole
haloacetamidines 6a-j..sup.a
##STR00063##
[0315] To a stirred solution of 4a-d, g (1.0 eq) in THF was added
phthalic anhydride (1.0 eq) and allowed to stir at rt under N.sub.2
for 18 h. Solvents were evaporated and the crude product was
purified by reverse phase HPLC using MeCN:H.sub.2O (0.5% TFA) as
the eluent to give the product in 78-86% yield. This product was
then treated with TFA to remove the Boc group giving the desired
2-CO.sub.2H-Bz-Orn-benzimidazole intermediate. The solvent was then
evaporated to dryness and the crude material was dried in vacuo. To
a stirred solution of the corresponding
2-CO.sub.2H-Bz-Orn-benzimidazole intermediate in dry MeOH was added
TEA (4.0 eq) followed by ethyl haloacetimidate HCl (2.0 eq). The
reaction was stirred under N.sub.2 at rt for 3 h. Solvents were
then evaporated under reduced pressure and the crude product was
purified by reverse phase HPLC using MeCN:H.sub.2O (0.5% TFA) as an
eluent to give compounds 6a-j in 52-69% yield.
(N1-(2-Carboxyl)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1H-benzo[d]imidazol--
2-yl)-L-ornithine) (6a)
##STR00064##
[0317] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.06 (dd, J=1.2
Hz, J=8.0 Hz, 2H), 7.84-7.79 (m, 2H), 7.71-7.67 (m, 1H), 7.63-7.56
(m, 4H), 5.59-5.54 (m, 1H), 5.26 (d, J=45.2 Hz, 2H), 3.52-3.48 (m,
2H), 2.38-2.23 (m, 2H), 2.09-1.89 (m, 2H). .sup.13C NMR (100 MHz,
CD.sub.3 OD) .delta. 173.5, 169.4, 164.8, 162.9, 162.6, 154.6,
139.0, 133.6, 133.3, 131.7, 131.2, 130.3, 128.9, 127.2, 115.2,
79.8, 78.1, 42.8, 30.5, 25.1. HRMS m/z calculated for
C.sub.21H.sub.23FN.sub.5O.sub.3(M+H.sup.+- H.sub.2O) 394.1674;
found 394.1673.
(N1-(2-Carboxyl)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1H-benzo[d]imidazol--
2-yl)-L-omithine) (6b)
##STR00065##
[0319] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.07 (dd, J=1.2
Hz, J=8.0 Hz, 2H), 7.83-7.79 (m, 2H), 7.72-7.67 (m, 1H), 7.64-7.57
(m, 4H), 5.57-5.54 (m, 1H), 4.41 (s, 2H), 3.50-3.46 (m, 2H),
2.39-2.24 (m, 2H), 2.09-1.88 (m, 2H). .sup.13C NMR (100 MHz,
CD.sub.3 OD) .delta. 173.5, 169.4, 164.8, 162.9, 162.6, 154.6,
139.0, 133.6, 133.3, 131.7, 131.2, 130.3, 128.9, 127.2, 115.2 (2C),
43.3, 40.1, 30.2 (2C), 24.9. HRMS m/z calculated for
C.sub.21H.sub.23ClN.sub.5O.sub.3(M+H.sup.+- H.sub.2O) 410.1378;
found 410.1382.
(N1-(2-Carboxyl)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-methyl-benzo[d]imi-
dazol-2-yl)-L-ornithine) (6c)
##STR00066##
[0321] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 7.99 (dd, J=1.4
Hz, J=7.8 Hz, 1H), 7.86 (dd, J=2.2 Hz, J=7.1 Hz, 1H), 7.80 (dd,
J=2.2 Hz, J=7.2 Hz, 1H), 7.66-7.54 (m, 4H), 7.49 (dd, J=1.4 Hz,
J=7.5 Hz, 1H), 5.68-5.64 (m, 1H), 5.28 (d, J=45.3 Hz, 2H), 4.19 (s,
3H), 3.52-3.46 (m, 2H), 2.41-2.31 (m, 1H), 2.29-2.21 (m, 1H),
2.09-1.99 (m, 1H), 1.96-1.86 (m, 1H). .sup.13C NMR (100 MHz,
CD.sub.3 OD) .delta. 171.7, 166.0, 160.6, 156.0, 151.9, 137.4,
131.8, 130.0, 129.7, 127.3, 125.7, 125.5, 114.9, 111.8, 110.5,
78.3, 76.6, 45.3, 41.3, 30.6, 28.3, 23.6. HRMS m/z calculated for
C.sub.22H.sub.24FN.sub.5O.sub.3(M+H.sup.+) 426.1936; found
426.1938.
(N1-(2-Carboxyl)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-methyl-benzo[d]imi-
dazol-2-yl)-L-ornithine) (6d)
##STR00067##
[0323] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 7.99 (dd, J=1.4
Hz, J=7.5 Hz, 1H), 7.83 (dd, J=2.3 Hz, J=7.2 Hz, 1H), 7.78 (dd,
J=1.8 Hz, J=7.2 Hz, 1H), 7.66-7.54 (m, 4H), 7.48 (dd, J=1.4 Hz,
J=7.4 Hz, 1H), 5.67-5.63 (m, 1H), 4.38 (s, 2H), 4.17 (s, 3H),
3.49-3.45 (m, 2H), 2.41-2.23 (m, 2H), 2.07-1.98 (m, 1H), 1.94-1.87
(m, 1H). .sup.13C NMR (100 MHz, CD.sub.3 OD) .delta. 171.1, 167.3,
161.9, 152.1, 137.5, 133.9, 133.7, 132.0, 130.2, 129.6, 127.3,
125.3, 115.2, 111.6, 109.9, 108.9, 101.1, 100.5, 45.3, 42.0, 30.6,
23.5. HRMS m/z calculated for
C.sub.22H.sub.24ClN.sub.5O.sub.3(M+H.sup.+) 442.1640; found
442.1642.
(N1-(2-Carboxyl)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-ethyl-benzo[d]imid-
azol-2-yl)-L-ornithine) (6e)
##STR00068##
[0325] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 7.99 (dd, J=1.6
Hz, J=7.4 Hz, 1H), 7.90 (dd, J=2.2 Hz, J=7.4 Hz, 1H), 7.82 (dd,
J=2.2 Hz, J=7.3 Hz, 1H), 7.66-7.54 (m, 4H), 7.48 (dd, J=1.6 Hz,
J=7.4 Hz, 1H), 5.66-5.62 (m, 1H), 5.28 (d, J=45.5 Hz, 2H),
4.70-4.61 (m, 2H), 3.52-3.41 (m, 2H), 2.40-2.20 (m, 2H), 2.09-1.98
(m, 1H), 1.94-1.83 (m, 1H), 1.59 (t, J=7.1 Hz, 3H). .sup.13C NMR
(100 MHz, CD.sub.3 OD) .delta. 171.3, 167.1, 160.1, 151.5, 137.2,
131.8, 129.8, 129.6, 128.8, 128.4, 126.8, 1260.0, 125.6, 125.6,
114.9, 111.9, 78.0, 45.1, 41.2, 40.2, 29.0, 23.4, 13.2. HRMS m/z
calculated for C.sub.23H.sub.26FN.sub.5O.sub.3(M+H.sup.+) 440.2092;
found 440.2092.
(N1-(2-Carboxyl)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-ethyl-benzo[d]imid-
azol-2-yl)-L-ornithine) (6f)
##STR00069##
[0327] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 7.99 (dd, J=1.3
Hz, J=7.4 Hz, 1H), 7.88 (dd, J=1.7 Hz, J=7.1 Hz, 1H), 7.81 (dd,
J=2.1 Hz, J=7.1 Hz, 1H), 7.65-7.54 (m, 4H), 7.47 (dd, J=1.7 Hz,
J=7.1 Hz, 1H), 5.65-5.61 (m, 1H), 4.68-4.59 (m, 2H), 4.37 (s, 2H),
3.47-3.41 (m, 2H), 2.40-2.21 (m, 2H), 2.09-1.98 (m, 1H), 1.95-1.85
(m, 1H), 1.60 (t, J=7.2 Hz, 3H). .sup.13C NMR (100 MHz, CD.sub.3
OD) .delta. 171.2, 167.0, 151.5, 137.3, 137.1, 132.1, 129.7, 129.1,
128.7, 127.8, 127.2, 125.3, 125.0, 115.1, 111.5, 87.0, 45.0, 41.7,
38.5, 28.5, 28.1, 23.3, 13.5. HRMS m/z calculated for
C.sub.23H.sub.26ClN.sub.5O.sub.3(M+H.sup.+) 456.1797; found
456.1799.
(N1-(2-Carboxyl)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-isopropyl-benzo[d]-
imidazol-2-yl)-L-ornithine) (62)
##STR00070##
[0329] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.05-8.02 (m,
1H), 7.99 (dd, J=1.7 Hz, J=7.6 Hz, 1H), 7.82-7.78 (m, 2H),
7.65-7.52 (m, 4H), 7.47 (dd, J=1.7 Hz, J=7.5 Hz, 1H), 5.67-5.63 (m,
1H), 5.27 (d, J=45.3 Hz, 2H), 5.31-5.25 (m, 1H), 3.53-3.43 (m, 2H),
2.37-2.20 (m, 2H), 2.07-1.96 (m, 1H), 1.93-1.85 (m, 1H), 1.82 (d,
J=2.4 Hz, 3H), 1.80 (d, J=2.3 Hz, 3H). .sup.13C NMR (100 MHz,
CD.sub.3 OD) .delta. 171.4, 167.5, 151.3, 137.1, 131.8, 130.1,
129.6, 129.1, 127.2, 125.9, 124.9, 115.7, 115.6, 114.1, 110.0,
78.3, 76.7, 50.5, 45.6, 41.4, 29.0, 23.5, 19.9, 19.8. HRMS m/z
calculated for C.sub.24H.sub.28FN.sub.5O.sub.3(M+H.sup.+) 454.2249;
found 454.2249.
(N1-(2-Carboxyl)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-isopropyl-benzo[d]-
imidazol-2-yl)-L-ornithine) (6h)
##STR00071##
[0331] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.10-8.07 (m,
1H), 7.99 (dd, J=1.7 Hz, J=7.4 Hz, 1H), 7.86-7.81 (m, 1H),
7.66-7.54 (m, 4H), 7.48 (dd, J=1.6 Hz, J=7.4 Hz, 1H), 5.70-5.66 (m,
1H), 5.37-5.30 (m, 1H), 4.37 (s, 2H), 3.47-3.43 (m, 2H), 2.39-2.19
(m, 2H), 2.09-1.98 (m, 1H), 1.93-1.82 (m, 1H), 1.84 (d, J=2.5 Hz,
3H), 1.82 (d, J=2.5 Hz, 3H). .sup.13C NMR (100 MHz, CD.sub.3 OD)
.delta. 171.6, 167.4, 163.2, 151.1, 136.9, 131.8, 130.4, 130.1,
129.5, 129.1, 127.2, 125.7, 125.3, 114.9, 114.5, 111.0, 50.8, 45.7,
41.7, 38.4, 28.9, 23.5, 19.8, 19.8. HRMS m/z calculated for
C.sub.24H.sub.28ClN.sub.5O.sub.3(M+H.sup.+) 470.1953; found
470.1959.
(N1-(2-Carboxyl)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(4-methoxy-1-methyl-b-
enzo[d]imidazol-2-yl)-L-ornithine) (6i)
##STR00072##
[0333] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 7.98 (dd, J=1.6
Hz, J=7.6 Hz, 1H), 7.65-7.61 (m, 1H), 7.57-7.47 (m, 3H), 7.40 (d,
J=8.2 Hz, 1H), 7.11 (d, J=8.2 Hz, 1H), 5.63-5.59 (m, 1H), 5.27 (d,
J=45.4 Hz, 2H), 4.17 (s, 3H), 4.05 (s, 3H), 3.50-3.45 (m, 2H),
2.40-2.30 (m, 1H), 2.29-2.18 (m, 1H), 2.08-1.96 (m, 1H), 1.89-1.77
(m, 1H). .sup.13C NMR (100 MHz, CD.sub.3 OD) .delta. 171.6, 166.7,
162.8, 160.9, 150.8, 148.2, 137.4, 134.5, 131.7, 129.8, 129.3,
128.6, 127.1, 126.6, 105.6, 103.4, 78.0, 76.2, 55.0, 45.5, 41.3,
30.5, 28.1, 23.2. HRMS m/z calculated for
C.sub.23H.sub.26FN.sub.5O.sub.4(M+H.sup.+) 456.2042; found
456.2039.
(N1-(2-Carboxyl)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(4-methoxy-1-methyl-b-
enzo[d]imidazol-2-yl)-L-ornithine) (6j)
##STR00073##
[0335] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 7.99 (dd, J=1.6
Hz, J=7.6 Hz, 1H), 7.65-7.61 (m, 1H), 7.58-7.47 (m, 3H), 7.39 (d,
J=8.3 Hz, 1H), 7.10 (d, J=8.1 Hz, 1H), 5.63-5.59 (m, 1H), 4.39 (s,
2H), 4.16 (s, 3H), 4.05 (s, 3H), 3.47-3.43 (m, 2H), 2.40-2.30 (m,
1H), 2.29-2.20 (m, 1H), 2.08-1.96 (m, 1H), 1.89-1.78 (m, 1H).
.sup.13C NMR (100 MHz, CD.sub.3 OD) .delta. 171.7, 167.2, 163.2,
150.9, 147.9, 137.2, 134.5, 132.0, 130.0, 129.6, 128.8, 127.2,
126.9, 115.0, 106.0, 103.6, 55.1, 45.6, 42.0, 38.4, 31.1, 28.0,
23.5. HRMS m/z calculated for
C.sub.23H.sub.26FN.sub.5O.sub.4(M+H.sup.+) 472.1745; found
472.1746.
3. General Procedure for Synthesis of Benzimidazole
haloacetamidines 7a-v..sup.a
##STR00074##
[0337] To a stirred solution of 4a-d, f-j (1.0 eq) in DMF was added
HOBt (2.0 eq), HBTU (2.0 eq), and DIPEA (3.0 eq) followed by
3-oxo-2,3-dihydro-1H-isoindole-4-carboxylic acid (1.0 eq) and
allowed to stir at rt for 12 h. The reaction mixture was then
diluted with water. The product was filtered, washed with water,
dried under vacuum, and obtained in 62-71% yield. This product was
then treated with 2 M HCl in Et.sub.2O to remove the Boc group
giving the Oxoisoindoline-Orn-benzimidazole intermediate. The
solvent was then evaporated to dryness and the crude material was
dried in vacuo. To a stirred solution of the corresponding
Oxoisoindoline-Om-benzimidazole intermediate in dry MeOH was added
TEA (4.0 eq) followed by ethyl haloacetimidate HCl (2.0 eq). The
reaction was stirred under N.sub.2 at rt for 3 h. Solvents were
then evaporated under reduced pressure and the crude product was
purified by reverse phase HPLC using MeCN:H.sub.2O (0.5% TFA) as an
eluent to give compounds 7a-v in 41-57% yield.
(N1-(3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1H-benzo[d]imi-
dazol-2-yl)-L-ornithine) (7a)
##STR00075##
[0339] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.18 (d, J=8.3
Hz, 1H), 7.83 (dd, J=1.6 Hz, J=8.3 Hz, 1H), 7.79-7.74 (m, 3H),
7.59-7.55 (m, 2H), 5.59-5.56 (m, 1H), 5.29 (d, J=45.3 Hz, 2H), 4.59
(s, 2H), 3.53-3.45 (m, 2H), 2.38-2.27 (m, 2H), 2.04-1.91 (m, 2H).
.sup.13C NMR (100 MHz, CD.sub.3 OD) .delta. 170.1, 166.6, 163.1,
154.3, 146.2, 131.8, 130.7, 130.2, 130.0, 128.8, 127.1, 126.9,
125.9, 118.0, 113.6, 102.5, 78.4, 76.7, 45.5, 41.2, 29.6, 23.7.
HRMS m/z calculated for C.sub.22H.sub.23FN.sub.6O.sub.2(M+H.sup.+)
423.1939; found 423.1941.
(N1-(3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1H-benzo[d]imi-
dazol-2-yl)-L-ornithine) (7b)
##STR00076##
[0341] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.20 (d, J=8.2
Hz, 1H), 7.83 (dd, J=1.6 Hz, J=7.7 Hz, 1H) 7.79-7.70 (m, 3H),
7.54-7.51 (m, 2H), 5.58-5.54 (m, 1H), 4.59 (s, 2H), 4.39 (s, 2H),
3.48-3.43 (m, 2H), 2.44-2.28 (m, 2H), 2.04-1.91 (m, 2H). .sup.13C
NMR (100 MHz, CD.sub.3 OD) .delta. 172.0, 163.3, 154.1, 146.1,
131.8, 131.5, 130.7, 130.2, 130.0, 128.8, 127.9, 127.1, 125.9,
125.8, 113.5, 110.0, 45.5, 43.1, 41.8, 38.8, 29.5, 23.6. HRMS m/z
calculated for C.sub.22H.sub.23ClN.sub.6O.sub.2(M+H.sup.+)
439.1644; found 439.1646.
(N1-(3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-methyl-benzo-
[d]imidazol-2-yl)-L-ornithine) (7c)
##STR00077##
[0343] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.18 (dd, J=1.3
Hz, J=7.5 Hz, 1H), 7.91 (dd, J=1.6 Hz, J=7.2 Hz, 1H), 7.82 (dd,
J=1.6 Hz, J=7.5 Hz, 1H), 7.75 (t, J=7.8 Hz, 2H), 7.67-7.58 (m, 2H),
5.65-5.62 (m, 1H), 5.30 (d, J=45.4 Hz, 2H), 4.58 (s, 2H), 4.21 (s,
3H), 3.54-3.44 (m, 2H), 2.37-2.25 (m, 2H), 2.13-2.03 (m, 1H),
2.02-1.93 (m, 1H). .sup.13C NMR (100 MHz, CD.sub.3 OD) .delta.
171.9, 166.2, 153.2, 146.3, 132.9, 131.7, 130.3, 130.1, 128.8,
127.2, 126.4, 126.0, 113.8, 112.3, 109.8, 78.5, 76.7, 45.4, 41.2,
37.3, 30.7, 28.6, 23.6. HRMS m/z calculated for
C.sub.23H.sub.25FN.sub.6O.sub.2(M+H.sup.+) 437.2096; found
437.2095.
(N1-(3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-methyl-benzo-
[d]imidazol-2-yl)-L-ornithine) (7d)
##STR00078##
[0345] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.18 (dd, J=1.3
Hz, J=7.6 Hz, 1H), 7.91 (dd, J=1.2 Hz, J=7.5 Hz, 1H), 7.82 (dd,
J=1.4 Hz, J=7.6 Hz, 1H), 7.74 (t, J=7.8 Hz, 2H), 7.66-7.58 (m, 2H),
5.66-5.62 (m, 1H), 4.58 (s, 2H), 4.41 (s, 2H), 4.21 (s, 3H),
3.51-3.44 (m, 2H), 2.37-2.26 (m, 2H), 2.13-1.93 (m, 2H). .sup.13C
NMR (100 MHz, CD.sub.3 OD) .delta. 171.9, 166.3, 163.4, 153.4,
146.3, 132.9, 131.8, 130.8, 130.3, 130.1, 128.9, 127.2, 126.4,
126.0, 114.0, 112.2, 46.7, 45.4, 41.8, 38.8, 30.7, 28.7, 23.6. HRMS
m/z calculated for C.sub.23H.sub.25ClN.sub.6O.sub.2(M+H.sup.+)
453.1800; found 453.1799.
(N1-(3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-ethyl-benzo[-
d]imidazol-2-yl)-L-ornithine) (7e)
##STR00079##
[0347] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.20 (dd, J=1.6
Hz, J=7.6 Hz, 1H), 7.94 (dd, J=1.6 Hz, J=7.4 Hz, 1H), 7.83 (dd,
J=1.2 Hz, J=7.6 Hz, 1H), 7.77-7.72 (m, 2H), 7.66-7.58 (m, 2H),
5.65-5.62 (m, 1H), 5.30 (d, J=45.4 Hz, 2H), 4.78-4.65 (m, 2H), 4.58
(s, 2H), 3.54-3.44 (m, 2H), 2.38-2.22 (m, 2H), 2.14-1.95 (m, 2H),
1.62 (t, J=7.4 Hz, 3H). .sup.13C NMR (100 MHz, CD.sub.3 OD) .delta.
171.9, 168.7, 165.9, 164.2, 156.4, 152.5, 145.9, 143.4, 131.6,
130.3, 130.1, 128.7, 127.7, 127.1, 126.3, 125.9, 114.1, 112.3,
45.3, 41.2, 40.4, 29.2, 23.8, 13.2. HRMS m/z calculated for
C.sub.24H.sub.27FN.sub.6O.sub.2(M+H.sup.+) 451.2252; found
451.2250.
(N1-(3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-ethyl-benzo[-
d]imidazol-2-yl)-L-ornithine) (7f)
##STR00080##
[0349] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.19 (dd, J=1.5
Hz, J=7.3 Hz, 1H), 7.94 (dd, J=1.5 Hz, J=7.5 Hz, 1H), 7.82 (dd,
J=1.3 Hz, J=7.3 Hz, 1H), 7.77-7.72 (m, 2H), 7.66-7.58 (m, 2H),
5.66-5.62 (m, 1H), 4.78-4.65 (m, 2H), 4.58 (s, 2H), 4.41 (s, 2H),
3.52-3.43 (m, 2H), 2.38-2.23 (m, 2H), 2.15-1.96 (m, 2H), 1.62 (t,
J=7.4 Hz, 3H). .sup.13C NMR (100 MHz, CD.sub.3 OD) .delta. 171.8,
166.1, 163.3, 159.2, 158.2, 153.0, 146.3, 131.8, 130.3, 128.8,
127.4, 126.3, 126.0, 116.5, 114.1, 112.4, 72.1, 45.4, 41.9, 40.5,
38.8, 29.2, 23.6, 13.3. HRMS m/z calculated for
C.sub.24H.sub.27ClN.sub.6O.sub.2(M+H.sup.+) 467.1957; found
467.1952.
(N1-(3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-isopropyl-be-
nzo[d]imidazol-2-yl)-L-omithine) (7g)
##STR00081##
[0351] .sup.1H NMR (CD.sub.3OD; 500 MHz): .delta. 8.21 (dd, J=1.5
Hz, J=7.7 Hz, 1H), 8.14 (dd, J=1.5 Hz, J=7.5 Hz, 1H), 7.85 (dd,
J=1.4 Hz, J=7.4 Hz, 1H), 7.80-7.76 (m, 2H), 7.66-7.61 (m, 2H),
5.73-5.70 (m, 1H), 5.37-5.31 (m, 1H), 5.34 (d, J=45.3 Hz, 2H), 4.61
(s, 2H), 3.60-3.49 (m, 2H), 2.40-2.22 (m, 2H), 2.15-2.07 (m, 1H),
2.06-1.99 (m, 1H), 1.88 (d, J=1.8 Hz, 3H), 1.86 (d, J=1.6 Hz, 3H).
.sup.13C NMR (125 MHz, CD.sub.3 OD) .delta. 171.8, 166.3, 163.2,
160.5, 160.1, 152.8, 146.4, 131.9, 131.3, 130.4, 130.3, 128.9,
127.2, 126.2, 125.7, 114.8, 114.3, 78.3, 77.0, 51.3, 45.5, 41.1,
29.3, 23.8, 19.5. HRMS m/z calculated for
C.sub.25H.sub.29FN.sub.6O.sub.2(M+H.sup.+) 465.2409; found
465.2409.
(N1-(3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-isopropyl-be-
nzo[d]imidazol-2-yl)-L-ornithine) (7h)
##STR00082##
[0353] .sup.1H NMR (CD.sub.3 OD; 500 MHz): .delta. 8.21 (dd, J=1.3
Hz, J=7.4, Hz, 1H), 8.14 (dd, J=2.0 Hz, J=7.6 Hz, 1H), 7.86 (dd,
J=1.6 Hz, J=7.3 Hz, 2H), 7.80-7.76 (m, 2H), 7.66-7.61 (m, 2H),
5.73-5.70 (m, 1H), 5.37-5.32 (m, 1H), 4.61 (s, 2H), 4.45 (s, 2H),
3.57-3.47 (m, 2H), 2.4-2.25 (m, 2H), 2.16-2.08 (m, 1H), 2.07-1.98
(m, 1H), 1.88 (d, J=3.6 Hz, 3H), 1.86 (d, J=3.5 Hz, 3H). .sup.13C
NMR (125 MHz, CD.sub.3 OD) .delta. 171.9, 166.3, 163.4, 160.6,
160.4, 152.7, 146.2, 131.9, 131.3, 130.3, 130.2, 128.9, 127.3,
126.2, 125.8, 114.7, 114.4, 51.3, 47.2, 45.4, 41.8, 38.7, 29.3,
23.7, 19.5. HRMS m/z calculated for
C.sub.25H.sub.29ClN.sub.6O.sub.2(M+H.sup.+) 481.2113; found
481.2112.
(N1-(3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(4-methoxy-1H-b-
enzo[d]imidazol-2-yl)-L-omithine) (7i)
##STR00083##
[0355] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.10 (dd, J=9.2
Hz, 1H), 7.73 (dd, J=9.3 Hz, 1H), 7.68-7.65 (m, 1H), 7.38-7.35 (m,
1H), 7.17 (dd, J=9.4 Hz, 1H), 6.98 (dd, J=9.2 Hz, 1H), 5.41-5.38
(m, 1H), 5.18 (d, J=45.5 Hz, 2H), 4.49 (s, 2H), 3.95 (s, 3H),
3.41-3.34 (m, 2H), 2.23-2.18 (m, 2H), 1.93-1.79 (m, 2H). .sup.13C
NMR (100 MHz, CD.sub.3 OD) .delta. 171.9, 166.5, 161.2, 153.6,
147.8, 146.1, 133.0, 131.8, 130.6, 130.2, 128.8, 127.1, 126.9,
105.9, 105.3, 100.0, 78.3, 76.9, 55.3, 45.5, 41.3, 29.9, 23.7. HRMS
m/z calculated for C.sub.23H.sub.25FN.sub.6O.sub.3(M+H.sup.+)
453.2045; found 453.2042.
(N1-(3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(4-methoxy-1H-b-
enzo[d]imidazol-2-yl)-L-ornithine) (7j)
##STR00084##
[0357] .sup.1H NMR (CD.sub.3 OD; 500 MHz) .delta. 8.11 (dd, J=1.1
Hz, J=7.8 Hz, 1H), 7.73 (dd, J=1.1 Hz, J=7.6 Hz, 1H), 7.67 (t,
J=7.8 Hz 1H), 7.36 (t, J=8.3 Hz, 1H), 7.17 (d, J=8.6 Hz, 1H), 6.97
(d, J=8.1 Hz, 1H), 5.41-5.38 (m, 1H), 4.49 (s, 2H), 4.29 (s, 2H),
3.95 (s, 3H), 3.39-3.33 (m, 2H), 2.23-2.19 (m, 2H), 1.93-1.80 (m,
2H). .sup.13C NMR (125 MHz, CD.sub.3 OD) .delta. 171.4, 166.1,
163.6, 154.1, 153.6, 146.1, 131.6, 130.7, 130.3, 129.6, 128.6,
127.2, 126.6, 105.8, 105.5, 100.0, 74.3, 55.1, 45.1, 41.8, 38.4,
29.2, 23.5. HRMS m/z calculated for
C.sub.23H.sub.25ClN.sub.6O.sub.3(M+H.sup.+) 469.1749; found
469.1750.
(N1-(3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(4-methoxy-1-me-
thyl-benzo[d]imidazol-2-yl)-L-ornithine)(7k)
##STR00085##
[0359] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.20 (dd, J=1.2
Hz, J=7.8 Hz, 1H), 7.81 (dd, J=1.3 Hz, J=7.6 Hz, 1H), 7.74 (t,
J=7.6 Hz, 1H), 7.53 (t, J=8.2 Hz, 1H), 7.41 (d, J=8.5 Hz, 1H), 7.10
(d, J=8.2 Hz), 5.60-5.56 (m, 1H), 5.30 (d, J=45.3 Hz, 2H), 4.56 (s,
2H), 4.16 (s, 3H), 4.00 (s, 3H), 3.54-3.43 (m, 2H), 2.40-2.23 (m,
2H), 2.12-2.01 (m, 1H), 1.98-1.87 (m, 1H). .sup.13C NMR (100 MHz,
CD.sub.3 OD) .delta. 171.8, 166.0, 160.8, 152.4, 148.1, 146.1,
134.3, 131.6, 130.3, 130.0, 128.9, 127.2, 126.8, 109.8, 106.2,
103.9, 78.3, 76.7, 55.3, 45.4, 41.2, 30.8, 28.8, 23.6. HRMS m/z
calculated for C.sub.24H.sub.27FN.sub.6O.sub.3 (M+H.sup.+)
467.2201; found 467.2201.
(N1-(3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(4-methoxy-1-me-
thyl-benzo[d]imidazol-2-yl)-L-ornithine)(7l)
##STR00086##
[0361] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.20 (dd, J=1.3
Hz, J=7.5 Hz, 1H), 7.77 (dd, J=1.3 Hz, J=7.5 Hz, 1H), 7.74 (t,
J=7.6 Hz, 1H), 7.53 (t, J=8.2 Hz, 1H), 7.40 (d, J=8.4 Hz, 1H), 7.09
(d, J=8.1 Hz, 1H), 5.61-5.57 (m, 1H), 4.56 (s, 2H), 4.40 (s, 2H),
4.15 (s, 3H), 4.00 (s, 3H), 3.48-3.44 (m, 2H), 2.40-2.23 (m, 2H),
2.11-2.01 (m, 1H), 1.98-1.87 (m, 1H). .sup.13C NMR (100 MHz,
CD.sub.3 OD) .delta. 171.8, 165.9, 163.4, 160.8, 152.4, 148.4,
146.2, 134.4, 132.8, 131.8, 130.4, 130.2, 128.9, 127.3, 126.9,
106.2, 103.7, 55.3, 45.4, 41.8, 38.7, 30.7, 28.7, 23.6. HRMS m/z
calculated for C.sub.24H.sub.27ClN.sub.6O.sub.3(M+H.sup.+)
483.1906; found 483.1907.
(N1-(3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(4-ethoxy-1-eth-
yl-benzo[d]imidazol-2-yl)-L-ornithine) (7m)
##STR00087##
[0363] .sup.1H NMR (CD.sub.3 OD; 50O MHz): .delta. 8.10 (dd, J=1.1
Hz, J=7.7 Hz, 1H), 7.71 (dd, J=1.1 Hz, J=7.7 Hz, 1H), 7.64 (t,
J=7.7 Hz, 1H), 7.41 (t, J=8.2 Hz, 1H), 7.30 (d, J=8.1 Hz, 1H), 6.97
(d, J=8.4 Hz, 1H), 5.50-5.47 (m, 1H), 5.21 (d, J=45.4 Hz, 2H), 4.46
(s, 2H), 4.18-4.13 (m, 2H), 4.08 (s, 3H), 3.44-3.36 (m, 2H),
2.32-2.23 (m, 1H), 2.22-2.14 (m, 1H), 2.02-1.94 (m, 1H), 1.89-1.79
(m, 1H), 1.36 (t, J=7.1 Hz, 3H). .sup.13C NMR (125 MHz, CD.sub.3
OD) .delta. 171.9, 166.0, 163.0, 161.3, 152.6, 147.2, 146.3, 134.3,
131.8, 130.5, 130.2, 128.9, 127.3, 127.1, 107.1, 103.7, 78.4, 76.9,
64.5, 45.4, 41.3, 31.0, 28.8, 23.8, 13.5. HRMS m/z calculated for
C.sub.25H.sub.29FN.sub.6O.sub.3(M+H.sup.+) 481.2358; found
481.2356.
(N1-(3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(4-ethoxy-1-eth-
yl-benzo[d]imidazol-2-yl)-L-ornithine) (7n)
##STR00088##
[0365] .sup.1H NMR (CD.sub.3 OD; 500 MHz): .delta. 8.08 (dd, J=1.2
Hz, J=7.7 Hz, 1H), 7.70 (dd, J=1.1 Hz, J=7.6 Hz, 1H), 7.63 (t,
J=7.7 Hz, 1H), 7.42 (t, J=8.3 Hz, 1H), 7.29 (d, J=8.1 Hz, 1H), 6.97
(d, J=8.1 Hz, 1H), 5.51-5.47 (m, 1H), 4.45 (s, 2H), 4.32 (s, 2H),
4.17-4.12 (m, 2H), 4.08 (s, 3H), 3.40-3.36 (m, 2H), 2.33-2.24 (m,
1H), 2.23-2.16 (m, 1H), 2.03-1.94 (m, 1H), 1.89-1.80 (m, 1H), 1.34
(t, J=7.1 Hz, 3H). .sup.13C NMR (125 MHz, CD.sub.3 OD) .delta.
171.9, 166.1, 163.5, 161.2, 160.8, 152.6, 147.1, 146.3, 134.3,
131.8, 130.4, 130.2, 129.0, 127.3, 121.5, 107.2, 103.7, 64.6, 45.4,
41.9, 38.8, 30.9, 28.8, 23.7, 13.4. HRMS m/z calculated for
C.sub.25H.sub.29ClN.sub.6O.sub.3(M+H.sup.+) 497.2062; found
497.2061.
(N1-(3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(5-methoxy-1-me-
thyl-benzo[d]imidazol-2-yl)-L-ornithine)(7o)
##STR00089##
[0367] .sup.1H NMR (CD.sub.3 OD; 500 MHz): .delta. 8.08 (dd, J=1.2
Hz, J=7.6 Hz, 1H), 7.68 (m, 3H), 7.15 (dd, J=2.4 Hz, J=9.2 Hz, 1H),
7.08 (d, J=2.4 Hz, 1H), 5.50-5.47 (m, 1H), 5.21 (d, J=45.2 Hz, 2H),
4.48 (s, 2H), 4.08 (s, 3H), 3.78 (s, 3H), 3.45-3.35 (m, 2H),
2.27-2.13 (m, 2H), 2.02-1.93 (m, 1H), 1.91-1.82 (m, 1H). .sup.13C
NMR (125 MHz, CD.sub.3 OD) .delta. 172.0, 166.3, 161.1, 159.5,
152.2, 146.3, 131.8, 131.5, 130.2, 128.9, 127.3, 127.0, 116.2,
113.0, 95.9, 78.3, 76.9, 55.2, 46.6, 45.5, 41.2, 30.7, 28.8, 23.8.
HRMS m/z calculated for C.sub.24H.sub.27FN.sub.6O.sub.3(M+H.sup.+)
467.2201; found 467.2201.
(N1-(3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(5-methoxy-1-me-
thyl-benzo[d]imidazol-2-yl)-L-ornithine) (.sup.7p)
##STR00090##
[0369] .sup.1H NMR (CD.sub.3 OD; 500 MHz): .delta. 8.08 (dd, J=1.1
Hz, J=7.5 Hz, 1H), 7.68 (m, 3H), 7.16 (dd, J=2.5 Hz, J=9.2 Hz, 1H),
7.08 (d, J=2.3 Hz, 1H), 5.51-5.48 (m, 1H), 4.48 (s, 2H), 4.08 (s,
3H), 3.78 (s, 3H), 3.43-3.33 (m, 2H), 2.28-2.14 (m, 2H), 2.01-1.93
(m, 1H), 1.91-1.82 (m, 1H). .sup.13C NMR (125 MHz, CD.sub.3 OD)
.delta. 171.9, 166.3, 163.5, 160.8, 159.4, 152.2, 146.3, 131.9,
131.5, 130.2, 128.9, 127.3, 127.0, 116.2, 113.0, 95.9, 55.2, 46.6,
45.5, 41.9, 38.7, 30.8, 28.7, 23.6. HRMS m/z calculated for
C.sub.24H.sub.27ClN.sub.6O.sub.3(M+H.sup.+) 483.1906; found
483.1906.
(N1-(3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(4-methoxy-1-et-
hyl-benzo[d]imidazol-2-yl)-L-ornithine) (7q)
##STR00091##
[0371] .sup.1H NMR (CD.sub.3OD; 500 MHz): .delta. 8.12 (dd, J=1.1
Hz, J=7.7 Hz, 1H), 7.72 (dd, J=1.1 Hz, J=7.6 Hz, 1H), 7.65 (t,
J=7.2 Hz, 1H), 7.44 (t, J=8.4 Hz, 1H), 7.34 (d, J=8.4 Hz, 1H), 7.00
(d, J=8.1 Hz, 1H), 5.52-5.48 (m, 1H), 5.21 (d, J=45.3 Hz, 2H),
4.64-4.57 (m, 1H), 4.56-4.50 (m, 1H), 4.48 (s, 2H), 3.92 (s, 3H),
3.45-3.35 (m, 2H), 2.29-2.21 (m, 1H), 2.20-2.13 (m, 1H), 2.03-1.94
(m, 1H), 1.90-1.83 (m, 1H), 1.49 (t, J=7.2 Hz, 3H). .sup.13C NMR
(125 MHz, CD.sub.3 OD) .delta. 172.0, 165.9, 161.1, 152.1, 148.2,
146.3, 133.3, 131.9, 130.5, 130.2, 128.9, 127.3, 127.0, 106.4,
104.0, 78.4, 77.0, 55.3, 46.8, 45.5, 41.4, 40.6, 29.4, 23.8, 13.4.
HRMS m/z calculated for C.sub.25H.sub.29FN.sub.6O.sub.3(M+H.sup.+)
481.2358; found 481.2353.
(N1-(3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(4-methoxy-1-et-
hyl-benzo[d]imidazol-2-yl)-L-ornithine) (7r)
##STR00092##
[0373] .sup.1H NMR (CD.sub.3 OD; 500 MHz): .delta. 8.11 (dd, J=1.3
Hz, J=7.9 Hz, 1H), 7.72 (dd, J=1.3 Hz, J=7.6 Hz, 1H), 7.64 (t,
J=7.6 Hz, 1H), 7.45 (t, J=8.3 Hz, 1H), 7.35 (d, J=8.4 Hz, 1H), 7.02
(d, J=8.2 Hz, 1H), 5.52-5.49 (m, 1H), 4.64-4.58 (m, 1H), 4.57-4.52
(m, 1H), 4.48 (s, 2H), 4.31 (s, 2H), 3.91 (s, 3H), 3.42-3.34 (m,
2H), 2.30-2.22 (m, 1H), 2.20-2.12 (m, 1H), 2.04-1.95 (m, 1H),
1.91-1.82 (m, 1H), 1.49 (t, J=7.5 Hz, 3H). .sup.13C NMR (125 MHz,
CD.sub.3 OD) .delta. 172.0, 166.0, 163.4, 160.7, 152.1, 148.1,
146.3, 133.1, 131.9, 130.5, 130.2, 128.9, 127.3, 121.9, 106.5,
104.0, 55.3, 46.8, 45.4, 41.8, 40.6, 38.8, 29.2, 23.7, 13.3. HRMS
m/z calculated for C.sub.25H.sub.29ClN.sub.6O.sub.3(M+H.sup.+)
497.2062; found 497.2060.
(N1-(3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(5-methoxy-1H-b-
enzo[d]imidazol-2-yl)-L-ornithine) (7s)
##STR00093##
[0375] .sup.1H NMR (CD.sub.3 OD; 500 MHz): .delta. 8.22 (d, J=8.3
Hz, 1H), 7.86 (d, J=8.3 Hz, 1H), 7.80 (t, J=6.6 Hz, 1H), 7.65 (d,
J=8.8 Hz, 1H), 7.23 (s, 1H), 7.20 (dd, J=2.3 Hz, J=8.8 Hz, 1H),
5.58-5.55 (m, 1H), 5.32 (d, J=45.7 Hz, 2H), 4.62 (s, 2H), 3.92 (s,
3H), 3.57-3.47 (m, 2H), 2.38-2.28 (m, 2H), 2.07-1.93 (m, 2H).
.sup.13C NMR (125 MHz, CD.sub.3 OD) .delta. 171.9, 169.3, 166.7,
159.0, 153.2, 146.2, 131.9, 130.8, 130.0, 128.9, 127.0, 115.9,
114.4, 99.8, 95.7, 78.3, 76.7, 55.2, 45.4, 41.2, 29.6, 23.7. HRMS
m/z calculated for C.sub.23H.sub.25FN.sub.6O.sub.3(M+H.sup.+)
453.2045; found 453.2037.
(N1-(3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(5-methoxy-1H-b-
enzo[d]imidazol-2-yl)-L-ornithine) (7t)
##STR00094##
[0377] .sup.1H NMR (CD.sub.3 OD; 500 MHz): .delta. 8.05 (d, J=8.1
Hz, 1H), 7.71 (d, J=8.2 Hz, 1H), 7.65 (t, J=7.6 Hz, 1H), 7.52 (d,
J=8.9 Hz, 1H), 7.11 (s, 1H), 7.07 (dd, J=2.2 Hz, J=8.4 Hz, 1H),
5.48-5.45 (m, 1H), 4.48 (s, 2H), 4.31 (s, 2H), 3.78 (s, 3H),
3.40-3.33 (m, 2H), 2.26-2.16 (m, 2H), 1.94-1.82 (m, 2H). .sup.13C
NMR (125 MHz, CD.sub.3 OD) .delta. 171.8, 166.8, 163.5, 159.2,
153.2, 146.1, 132.2, 131.9, 130.8, 130.0, 128.8, 127.0, 125.2,
116.2, 114.4, 95.8, 55.0, 45.4, 41.8, 38.8, 29.6, 23.5. HRMS m/z
calculated for C.sub.23H.sub.25ClN.sub.6O.sub.3(M+H.sup.+)
469.1749; found 469.1741.
(N1-(3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(5-methoxy-1-et-
hyl-benzo[d]imidazol-2-yl)-L-ornithine) (7u)
##STR00095##
[0379] .sup.1H NMR (CD.sub.3 OD; 500 MHz): .delta. 8.20 (d, J=7.7
Hz, 1H), 7.84 (d, J=8.4 Hz, 2H), 7.77 (t, J=7.7 Hz, 1H), 7.26 (dd,
J=2.2 Hz, J=8.4 Hz, 1H), 7.22 (d, J=2.3 Hz, 1H), 5.64-5.61 (m, 1H),
5.34 (d, J=45.5 Hz, 2H), 4.78-4.64 (m, 2H), 4.61 (s, 2H), 3.90 (s,
3H), 3.59-3.48 (m, 2H), 2.41-2.24 (m, 2H), 2.19-1.98 (m, 2H),
1.84-1.80 (m, 1H), 1.75-1.69 (m, 1H), 1.64 (t, J=7.2 Hz, 3H).
.sup.13C NMR (125 MHz, CD.sub.3 OD) .delta. 171.9, 166.2, 163.2,
161.1, 159.4, 151.8, 146.3, 131.8, 130.4, 128.9, 127.2, 125.7,
116.2, 113.3, 96.1, 78.3, 77.0, 55.0, 46.7, 45.5, 41.4, 40.7, 29.4,
23.7, 13.4. HRMS m/z calculated for
C.sub.25H.sub.29FN.sub.6O.sub.3(M+H.sup.+) 481.2358; found
481.2358.
(N1-(3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(5-methoxy-1-et-
hyl-benzo[d]imidazol-2-yl)-L-ornithine) (7v)
##STR00096##
[0381] .sup.1H NMR (CD.sub.3 OD; 500 MHz): .delta. 8.07 (d, J=8.0
Hz, 1H), 7.71 (d, J=8.6 Hz, 2H), 7.64 (t, J=7.9 Hz, 1H), 7.13 (dd,
J=2.6 Hz, J=8.8 Hz, 1H), 7.08 (d, J=2.3 Hz, 1H), 5.51-5.48 (m, 1H),
4.64-4.50 (m, 2H), 4.47 (s, 2H), 4.33 (s, 2H), 3.77 (s, 3H),
3.42-3.35 (m, 2H), 2.28-2.11 (m, 2H), 2.03-1.83 (m, 2H), 1.72-1.67
(m, 1H), 1.62-1.58 (m, 1H), 1.51 (t, J=8.1 Hz, 3H). .sup.13C NMR
(125 MHz, CD.sub.3 OD) .delta. 171.9, 166.2, 163.4, 161.2, 161.0,
159.5, 151.9, 146.3, 131.8, 130.3, 128.8, 127.2, 125.8, 125.6,
116.2, 113.2, 96.0, 55.2, 46.6, 45.5, 41.8, 40.6, 38.8, 29.4, 23.7,
13.4. HRMS m/z calculated for
C.sub.25H.sub.29ClN.sub.6O.sub.3(M+H.sup.+) 497.2062; found
497.2065.
5. General Procedure for Synthesis of Benzimidazole
haloacetamidines 8a-b'..sup.a
##STR00097##
[0383] To a stirred solution of 4a-d, f-j (1.0 eq) in DMF was added
HOBt (2.0 eq), HBTU (2.0 eq), and DIPEA (3.0 eq) followed by
2-alkyl-3-oxo-4-isoindoline carboxylic acid (1.0 eq) and allowed to
stir at rt for 12 h. The reaction mixture was then diluted with
water. The product was filtered, washed with water, dried under
vacuum, and obtained in 62-71% yield. This product was then treated
with 2 M HCl in Et.sub.2O to remove the Boc group giving the
N-alkyl-Oxoisoindoline-Orn-benzimidazole intermediate. The solvent
was then evaporated to dryness and the crude material was dried in
vacuo. To a stirred solution of the corresponding
N-alkyl-Oxoisoindoline-Om-benzimidazole intermediate in dry MeOH
was added TEA (4.0 eq) followed by ethyl haloacetimidate HCl (2.0
eq). The reaction was stirred under N.sub.2 at rt for 3 h. Solvents
were then evaporated under reduced pressure and the crude product
was purified by reverse phase HPLC using MeCN:H.sub.2O (0.5% TFA)
as an eluent to give compounds 8a-b' in 56-74% yield.
(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1H-ben-
zo[d]imidazol-2-yl)-L-ornithine) (8a)
##STR00098##
[0385] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.11 (dd, J=1.1
Hz, J=7.7 Hz, 1H), 7.80-7.70 (m, 4H), 7.60-7.55 (m, 2H), 5.64-5.59
(m, 1H), 5.28 (d, J=45.4 Hz, 2H), 4.63 (s, 2H), 3.80-3.74 (m, 2H),
3.52-3.48 (m, 2H), 2.39-2.28 (m, 2H), 2.06-1.92 (m, 2H), 1.35 (t,
J=7.3 Hz, 3H). .sup.13C NMR (100 MHz, CD.sub.3 OD) .delta. 168.3,
166.4, 163.1, 162.9, 160.7, 154.0, 143.3, 131.3, 131.1, 130.2,
129.8, 129.1, 126.4, 126.1 (2C), 113.5, 78.3, 76.6, 49.2, 41.2,
37.5, 29.5, 23.7, 11.9. HRMS m/z calculated for
C.sub.24H.sub.27FN.sub.6O.sub.2(M+H.sup.+) 451.2252; found
451.2249.
(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1H-ben-
zo[d]imidazol-2-yl)-L-ornithine) (8b)
##STR00099##
[0387] .sup.1H NMR (CD.sub.3OD; 400 MHz): .delta. 8.12 (dd, J=1.2
Hz, J=7.8 Hz, 1H), 7.81-7.70 (m, 4H), 7.60-7.56 (m, 2H), 5.63-5.59
(m, 1H), 4.63 (s, 2H), 4.38 (s, 2H), 3.80-3.75 (m, 2H), 3.50-3.45
(m, 2H), 2.41-2.31 (m, 2H), 2.08-1.92 (m, 2H), 1.35 (t, J=7.2 Hz,
3H). .sup.13C NMR (100 MHz, CD.sub.3 OD) .delta. 168.3, 166.5,
163.2, 161.1, 160.8, 154.1, 143.4, 131.3, 131.1, 130.2, 129.8,
129.0, 126.5, 125.9 (2C), 113.5 (2C), 49.2, 41.7, 38.7, 37.4, 29.4,
23.4, 12.0. HRMS m/z calculated for
C.sub.24H.sub.27ClN.sub.6O.sub.2(M+H.sup.+) 467.1957; found
467.1954.
(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-meth-
yl-benzo[d]imidazol-2-yl)-L-ornithine) (8c)
##STR00100##
[0389] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.17 (dd, J=1.2
Hz, J=7.9 Hz, 1H), 7.88 (dd, J=1.5 Hz, J=7.7 Hz, 1H), 7.80 (dd,
J=1.2 Hz, J=7.8 Hz, 1H), 7.74-7.70 (m, 2H), 7.64-7.56 (m, 2H),
5.67-5.63 (m, 1H), 5.28 (d, J=45.5 Hz, 2H), 4.64 (s, 2H), 4.19 (s,
3H), 3.79-3.72 (m, 2H), 3.56-3.45 (m, 2H), 2.41-2.25 (m, 2H),
2.14-2.03 (m, 1H), 2.01-1.89 (m, 1H), 1.34 (t, J=7.5 Hz, 3H).
.sup.13C NMR (100 MHz, CD.sub.3 OD) .delta. 168.1, 166.0, 162.9,
158.3, 152.8, 143.4, 133.1, 131.3, 130.3, 129.8, 129.3, 126.8,
126.1, 125.6, 114.1, 111.9, 109.8, 78.4, 76.5, 49.2, 41.3, 37.5,
30.5, 28.8, 23.6, 12.0. HRMS m/z calculated for
C.sub.25H.sub.29FN.sub.6O.sub.2(M+H.sup.+) 465.2409; found
465.2404.
(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-meth-
yl-benzo[d]imidazol-2-yl)-L-ornithine) (8d)
##STR00101##
[0391] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.16 (dd, J=1.5
Hz, J=7.7 Hz, 1H), 7.91 (dd, J=1.1 Hz, J=7.6 Hz, 1H), 7.80 (dd,
J=1.5 Hz, J=7.6 Hz, 1H), 7.75-7.70 (m, 2H), 7.66-7.58 (m, 2H),
5.68-5.63 (m, 1H), 4.64 (s, 2H), 4.38 (s, 2H), 4.21 (s, 3H),
3.79-3.73 (m, 2H), 3.50-3.46 (m, 2H), 2.42-2.26 (m, 2H), 2.16-2.05
(m, 1H), 2.02-1.91 (m, 1H), 1.34 (t, J=7.2 Hz, 3H). .sup.13C NMR
(100 MHz, CD.sub.3 OD) .delta. 168.4, 165.9, 163.2, 160.8, 153.3,
143.5, 132.5, 131.2, 130.1, 129.5, 129.2, 126.7, 126.3, 125.7,
113.9, 112.1, 109.9, 49.2, 41.7, 38.7, 37.5, 30.6, 28.7, 23.4,
11.9. HRMS m/z calculated for
C.sub.25H.sub.29ClN.sub.6O.sub.2(M+H.sup.+) 481.2113; found
481.2118.
(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-ethy-
l-benzo[d]imidazol-2-yl)-L-ornithine) (8e)
##STR00102##
[0393] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.15 (dd, J=1.1
Hz, J=7.8 Hz, 1H), 7.95 (dd, J=1.2 Hz, J=7.2 Hz, 1H), 7.80 (dd,
J=1.2 Hz, J=7.7 Hz, 1H), 7.76-7.69 (m, 2H), 7.67-7.59 (m, 2H),
5.67-5.63 (m, 1H), 5.29 (d, J=45.4 Hz, 2H), 4.82-4.66 (m, 2H), 4.64
(s, 2H), 3.81-3.72 (m, 3H), 3.56-3.46 (m, 2H), 2.42-2.23 (m, 2H),
2.18-2.08 (m, 1H), 2.05-1.93 (m, 1H), 1.64 (t, J=7.2 Hz, 3H), 1.35
(t, J=7.3 Hz, 3H). .sup.13C NMR (100 MHz, CD.sub.3 OD) .delta.
168.1, 165.9, 163.0, 153.1, 143.5, 131.6, 131.3, 130.6, 130.1,
129.6, 129.2, 126.7, 126.4, 126.1, 113.9, 112.5, 78.4, 76.7, 49.2,
41.2, 40.6, 37.6, 29.2, 23.8, 13.2, 12.1. HRMS m/z calculated for
C.sub.26H.sub.31FN.sub.6O.sub.2(M+H.sup.+) 479.2565; found
479.2565.
(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-ethy-
l-benzo[d]imidazol-2-yl)-L-ornithine) (8f)
##STR00103##
[0395] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.15 (dd, J=1.2
Hz, J=7.8 Hz, 1H), 7.94 (dd, J=1.1 Hz, J=7.1 Hz, 1H), 7.80 (dd,
J=1.1 Hz, 7.4 Hz, 1H), 7.76-7.69 (m, 2H), 7.66-7.58 (m, 2H),
5.68-5.64 (m, 1H), 4.81-4.66 (m, 2H), 4.63 (s, 2H), 4.40 (s, 2H),
3.79-3.74 (m, 3H), 3.52-3.47 (m, 2H), 2.43-2.33 (m, 1H), 2.31-2.25
(m, 1H), 2.18-2.07 (m, 1H), 2.05-1.94 (m, 1H), 1.63 (t, J=7.3 Hz,
3H), 1.34 (t, J=7.3 Hz, 3H). .sup.13C NMR (100 MHz, CD.sub.3 OD)
.delta. 168.3, 166.3, 163.1, 161.2, 152.8, 143.8, 131.6, 131.2,
130.5, 130.0, 129.6, 129.0, 126.7, 126.3, 125.7, 114.0, 112.2,
49.2, 42.0, 40.4, 38.5, 37.4, 29.0, 23.3, 13.3, 11.9. HRMS m/z
calculated for C.sub.26H.sub.31ClN.sub.6O.sub.2(M+H.sup.+)
495.2270; found 495.2273.
(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-isop-
ropyl-benzo[d]imidazol-2-yl)-L-ornithine) (8g)
##STR00104##
[0397] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.16-8.10 (m,
2H), 7.81-7.69 (m, 3H), 7.62-7.57 (m, 2H), 5.72-5.68 (m, 1H),
5.34-5.29 (m, 1H), 5.28 (d, J=45.4 Hz, 2H), 4.64 (s, 2H), 3.80-3.73
(m, 2H), 3.56-3.47 (m, 2H), 2.42-2.21 (m, 2H), 2.16-2.05 (m, 1H),
2.03-1.92 (m, 1H), 1.85 (d, J=2.7 Hz, 3H), 1.83 (d, J=2.7 Hz, 3H),
1.35 (t, J=7.3 Hz, 3H). .sup.13C NMR (100 MHz, CD.sub.3 OD) .delta.
168.4, 166.1, 163.2, 162.7, 161.2, 160.8, 152.7, 143.6, 131.4,
130.1, 129.7, 129.1, 126.6, 126.0, 125.6, 114.7, 114.3, 78.4, 76.6,
51.2, 49.2, 41.3, 37.4, 29.3, 23.8, 19.4, 12.0. HRMS m/z calculated
for C.sub.27H.sub.33FN.sub.6O.sub.2(M+H.sup.+) 493.2722; found
493.2723.
(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-isop-
ropyl-benzo[d]imidazol-2-yl)-L-ornithine) (8h)
##STR00105##
[0399] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.16-8.09 (m,
2H), 7.81-7.69 (m, 3H), 7.61-7.57 (m, 2H), 5.72-5.68 (m, 1H),
5.36-5.29 (m, 1H), 4.64 (s, 2H), 4.40 (s, 2H), 3.79-3.74 (m, 2H),
3.52-3.48 (m, 2H), 2.42-2.22 (m, 2H), 2.17-2.06 (m, 1H), 2.05-1.92
(m, 1H), 1.85 (d, J=3.4 Hz, 3H), 1.83 (d, J=3.4 Hz, 3H), 1.34 (t,
J=7.2 Hz, 3H). .sup.13C NMR (100 MHz, CD.sub.3 OD) .delta. 168.5,
166.3, 163.4, 161.2, 160.9, 160.5, 152.8, 143.6, 131.3, 130.2,
129.8, 129.2, 126.8, 126.2, 125.7, 115.0, 114.7, 114.3, 51.3, 49.3,
41.9, 38.7, 37.5, 29.4, 23.6, 19.6, 12.1. HRMS m/z calculated for
C.sub.27H.sub.33ClN.sub.6O.sub.2(M+H.sup.+) 509.2426; found
509.2426.
(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(4-meth-
oxy-1H-benzo[d]imidazol-2-yl)-L-ornithine) (8i)
##STR00106##
[0401] .sup.1H NMR (CD.sub.3 OD; 500 MHz): .delta. 8.04 (d, J=7.8
Hz, 1H), 7.70 (d, J=7.9 Hz, 1H), 7.63 (t, J=8.7 Hz, 1H), 7.39 (t,
J=7.8 Hz, 1H), 7.19 (d, J=8.7 Hz, 1H), 6.99 (d, J=8.7 Hz, 1H),
5.45-5.42 (m, 1H), 5.18 (d, J=45.3 Hz, 2H), 4.55 (s, 2H), 3.95 (s,
3H), 3.71-3.64 (m, 2H), 3.43-3.37 (m, 2H), 2.26-2.21 (m, 2H),
1.96-1.79 (m, 2H), 1.26 (t, J=7.9 Hz, 3H). .sup.13C NMR (125 MHz,
CD.sub.3 OD) .delta. 168.6, 166.7, 163.2, 153.5, 147.7, 143.6,
132.6, 131.4, 130.4, 130.0, 129.2, 127.2, 126.6, 122.0, 106.2,
105.4, 78.3, 76.8, 55.3, 49.4, 41.4, 37.5, 29.7, 23.8, 12.1. HRMS
m/z calculated for C.sub.25H.sub.29FN.sub.6O.sub.3(M+H) 481.2358;
found 481.2358.
(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(4-meth-
oxy-1H-benzo[d]imidazol-2-yl)-L-ornithine) (8j)
##STR00107##
[0403] .sup.1H NMR (CD.sub.3 OD; 500 MHz): .delta. 8.16 (d, J=7.4
Hz, 1H), 7.83 (d, J=7.2 Hz, 1H), 7.76 (d, J=7.8 Hz, 1H), 7.52 (t,
J=7.5 Hz, 1H), 7.32 (d, J=8.1 Hz, 1H), 7.12 (d, J=7.8 Hz, 1H),
5.58-5.55 (m, 1H), 4.67 (s, 2H), 4.41 (s, 2H), 4.07 (s, 3H),
3.83-3.79 (m, 2H), 3.52-3.48 (m, 2H), 2.39-2.34 (m, 2H), 2.09-1.93
(m, 2H), 1.38 (t, J=7.5 Hz, 3H). .sup.13C NMR (125 MHz, CD.sub.3
OD) .delta. 168.6, 166.5, 163.5, 153.7, 147.6, 143.5, 132.6, 131.3,
130.4, 129.9, 129.1, 127.3, 126.7, 125.6, 121.9, 106.2, 105.2,
55.3, 49.4, 41.9, 38.8, 37.6, 29.7, 23.6, 11.9. HRMS m/z calculated
for C.sub.25H.sub.29ClN.sub.6O.sub.3(M+H.sup.+) 497.2062; found
497.2060.
(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(4-meth-
oxy-1-methyl-benzo[d]imidazol-2-yl)-L-ornithine) (8k)
##STR00108##
[0405] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.16 (dd, J=1.4
Hz, J=7.8 Hz, 1H), 7.80 (dd, J=1.3 Hz, J=7.8 Hz, 1H), 7.71 (t,
J=7.5 Hz, 1H), 7.55 (t, J=8.3 Hz, 1H), 7.42 (dd, J=1.1 Hz, J=8.6
Hz, 1H), 7.11 (d, J=8.2 Hz, 1H), 5.62-5.58 (m, 1H), 5.19 (d, J=45.5
Hz, 2H), 4.63 (s, 2H), 4.18 (s, 3H), 4.01 (s, 3H), 3.81-3.70 (m,
2H), 3.52-3.48 (m, 2H), 2.43-2.24 (m, 2H), 2.14-2.03 (m, 1H),
1.98-1.86 (m, 1H), 1.34 (t, J=7.1 Hz, 3H). .sup.13C NMR (100 MHz,
CD.sub.3 OD) .delta. 168.5, 166.1, 160.7, 152.5, 147.9, 143.7,
134.4, 131.3, 130.3, 129.7, 129.3, 127.1, 126.7, 115.0, 106.5,
103.9, 78.4, 76.6, 55.3, 49.2, 41.3, 37.6, 30.9, 28.8, 23.8, 12.0.
HRMS m/z calculated for C.sub.26H.sub.31FN.sub.6O.sub.3(M+H.sup.+)
495.2514; found 495.2512.
(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(4-meth-
oxy-1-methyl-benzo[d]imidazol-2-yl)-L-ornithine) (8l)
##STR00109##
[0407] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.17 (dd, J=1.1
Hz, J=7.8 Hz, 1H), 7.79 (dd, J=1.3 Hz, J=7.9 Hz, 1H), 7.72 (t,
J=7.6 Hz, 1H), 7.56 (t, J=8.4 Hz, 1H), 7.43 (dd, J=1.1 Hz, J=8.7
Hz, 1H), 7.12 (d, J=8.1 Hz, 1H), 5.62-5.59 (m, 1H), 4.63 (s, 2H),
4.39 (s, 2H), 4.19 (s, 3H), 4.01 (s, 3H), 3.82-3.71 (m, 2H),
3.50-3.46 (m, 2H), 2.44-2.26 (m, 2H), 2.15-2.05 (m, 1H), 2.00-1.87
(m, 1H), 1.34 (t, J=7.3 Hz, 3H). .sup.13C NMR (100 MHz, CD.sub.3
OD) .delta. 168.4, 166.1, 163.3, 152.5, 148.4, 147.8, 143.7, 134.2,
131.4, 130.2, 129.9, 129.2, 127.2, 126.8, 106.5, 103.8, 55.2, 49.4,
46.8, 41.9, 38.6, 37.5, 30.9, 28.7, 23.6, 11.9. HRMS m/z calculated
for C.sub.26H.sub.31ClN.sub.6O.sub.3(M+H.sup.+) 511.2219; found
511.2220.
(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(4-etho-
xy-1-methyl-benzo[d]imidazol-2-yl)-L-ornithine) (8m)
##STR00110##
[0409] .sup.1H NMR (CD.sub.3 OD; 500 MHz): .delta. 8.06 (dd, J=1.1
Hz, J=8.1 Hz, 1H), 7.69 (dd, J=1.2 Hz, J=7.6 Hz, 1H), 7.60 (t,
J=7.5 Hz, 1H), 7.42 (t, J=8.4 Hz, 1H), 7.30 (dd, J=1.0 Hz, J=8.2
Hz, 1H), 6.98 (d, J=8.2 Hz, 1H), 5.52-5.48 (m, 1H), 5.18 (d, J=45.6
Hz, 2H), 4.52 (s, 2H), 4.19-4.14 (m, 2H), 4.08 (s, 3H), 3.70-3.62
(m, 2H), 3.43-3.39 (m, 2H), 2.35-2.26 (m, 1H), 2.24-2.16 (m, 1H),
2.04-1.95 (m, 1H), 1.87-1.78 (m, 1H), 1.36 (t, J=7.1 Hz, 3H), 1.25
(t, J=7.1 Hz, 3H). .sup.13C NMR (125 MHz, CD.sub.3 OD) .delta.
168.5, 166.2, 152.5, 147.2, 143.8, 134.3, 131.4, 130.3, 129.8,
129.4, 127.1, 126.7, 121.6, 107.2, 103.7, 78.3, 76.9, 64.5, 49.3,
46.8, 41.5, 37.5, 30.9, 28.8, 23.8, 13.4, 12.0. HRMS m/z calculated
for C.sub.27H.sub.33FN.sub.6O.sub.3(M+H.sup.+) 509.2671; found
509.2676.
(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2chloro-1-iminoethyl)-1-(4-ethox-
y-1-methyl-benzo[d]imidazol-2-yl)-L-ornithine)(8n)
##STR00111##
[0411] .sup.1H NMR (CD.sub.3 OD; 500 MHz): .delta. 8.05 (dd, J=1.1
Hz, J=8.0 Hz, 1H), 7.68 (dd, J=1.1 Hz, J=7.6 Hz, 1H), 7.60 (t,
J=7.8 Hz, 1H), 7.41 (t, J=8.2 Hz, 1H), 7.28 (dd, J=1.0 Hz, J=8.6
Hz, 1H), 6.96 (d, J=8.2 Hz, 1H), 5.52-5.49 (m, 1H), 4.51 (s, 2H),
4.30 (s, 2H), 4.17-4.12 (m, 2H), 4.08 (s, 3H), 3.68-3.64 (m, 2H),
3.40-3.37 (m, 2H), 2.35-2.26 (m, 1H), 2.25-2.18 (m, 1H), 2.05-1.95
(m, 1H), 1.87-1.78 (m, 1H), 1.35 (t, J=7.2 Hz, 3H), 1.25 (t, J=7.3
Hz, 3H). .sup.13C NMR (125 MHz, CD.sub.3 OD) .delta. 168.5, 166.2,
163.3, 152.6, 147.3, 143.6, 134.4, 131.4, 130.3, 129.8, 129.3,
127.0, 126.6, 121.9, 107.2, 103.7, 64.6, 49.2, 46.9, 42.0, 38.7,
37.5, 30.9, 28.9, 23.7, 13.5, 12.1. HRMS m/z calculated for
C.sub.27H.sub.33ClN.sub.6O.sub.3(M+H.sup.+) 525.2375; found
525.2378.
(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(5-meth-
oxy-1-methyl-benzo[d]imidazol-2-yl)-L-ornithine) (8o)
##STR00112##
[0413] .sup.1H NMR (CD.sub.3 OD; 500 MHz): .delta. 8.06 (dd, J=1.2
Hz, J=7.9 Hz, 1H), 7.70 (m, 2H), 7.63-7.59 (t, J=7.9 Hz, 1H), 7.14
(dd, J=2.4 Hz, J=9.2 Hz, 1H), 7.09 (d, J=2.3 Hz, 1H), 5.52-5.49 (m,
1H), 5.18 (d, J=45.3 Hz, 2H), 4.53 (s, 2H), 4.09 (s, 3H), 3.77 (s,
3H), 3.69-3.64 (m, 2H), 3.47-3.37 (m, 2H), 2.31-2.16 (m, 2H),
2.04-1.95 (m, 1H), 1.90-1.81 (m, 1H), 1.25 (t, J=7.4 Hz, 3H).
.sup.13C NMR (125 MHz, CD.sub.3 OD) .delta. 168.6, 166.2, 161.0,
159.6, 152.2, 143.7, 131.4, 130.3, 129.8, 129.3, 127.0, 126.7,
116.3, 113.1, 95.9, 78.3, 76.9, 55.1, 49.3, 46.7, 41.4, 37.5, 30.9,
28.7, 23.8, 12.0. HRMS m/z calculated for
C.sub.26H.sub.31FN.sub.6O.sub.3(M+H.sup.+) 495.2514; found
495.2514.
(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(5-meth-
oxy-1-methyl-benzo[d]imidazol-2-yl)-L-ornithine) (8p)
##STR00113##
[0415] .sup.1H NMR (CD.sub.3 OD; 500 MHz): .delta. 8.05 (dd, J=1.1
Hz, J=7.7 Hz, 1H), 7.70-7.67 (m, 2H), 7.61 (t, J=7.6 Hz, 1H), 7.13
(dd, J=2.5 Hz, J=9.1 Hz, 1H), 7.09 (d, J=2.4 Hz, 1H), 5.52-5.49 (m,
2H), 4.52 (s, 2H), 4.30 (s, 3H), 4.09 (s, 3H), 3.77 (s, 3H),
3.69-3.63 (m, 2H), 3.41-3.38 (m, 2H), 2.32-2.16 (m, 2H), 2.05-1.96
(m, 1H), 1.91-1.82 (m, 1H), 1.24 (t, J=7.2 Hz, 3H). .sup.13C NMR
(125 MHz, CD.sub.3 OD) .delta. 168.6, 166.3, 163.4, 160.8, 152.2,
143.7, 131.4, 130.2, 129.8, 129.3, 127.0, 126.7, 125.7, 116.2,
113.1, 95.9, 55.2, 49.3, 46.8, 41.9, 38.7, 37.5, 30.9, 28.8, 23.6,
12.0. HRMS m/z calculated for
C.sub.26H.sub.31ClN.sub.6O.sub.3(M+H.sup.+) 511.2219; found
511.2218.
(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(4-meth-
oxy-1-isopropyl-benzo[d]imidazol-2-yl)-L-ornithine) (8q)
##STR00114##
[0417] .sup.1H NMR (CD.sub.3 OD; 500 MHz): .delta. 8.06 (d, J=7.9
Hz, 1H), 7.70 (d, J=8.0 Hz, 1H), 7.61 (t, J=8.2 Hz, 1H), 7.44 (t,
J=8.2 Hz, 1H), 7.31 (d, J=8.2 Hz, 1H), 7.01 (d, J=8.0 Hz, 1H),
5.52-5.49 (m, 1H), 5.18 (d, J=45.2 Hz, 2H), 4.57-4.51 (m, 1H), 4.49
(s, 2H), 4.07 (s, 3H), 3.91 (s, 3H), 3.44-3.37 (m, 2H), 2.33-2.25
(m, 1H), 2.24-2.17 (m, 1H), 2.04-1.95 (m, 1H), 1.87-1.78 (m, 1H),
1.29 (d, J=2.8, 3H), 1.28 (d, J=2.7 Hz, 3H). .sup.13C NMR (125 MHz,
CD.sub.3 OD) .delta. 168.2, 166.1, 152.6, 148.0, 143.8, 134.5,
131.3, 130.3, 129.9, 129.5, 127.1, 126.8, 121.9, 106.5, 103.9,
78.3, 76.9, 55.3, 46.8, 45.4, 44.1, 41.4, 30.9, 28.8, 23.8, 19.3,
19.2. HRMS m/z calculated for
C.sub.27H.sub.33FN.sub.6O.sub.3(M+H.sup.+) 509.2669; found
509.2674.
(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(4-meth-
oxy-1-isopropyl-benzo[d]imidazol-2-yl)-L-ornithine)(8r)
##STR00115##
[0419] .sup.1H NMR (CD.sub.3 OD; 500 MHz): .delta. 8.03 (d, J=8.1
Hz, 1H), 7.68 (d, J=7.7 Hz, 1H), 7.58 (t, J=7.8 Hz, 1H), 7.43 (t,
J=7.9 Hz, 1H), 7.31 (d, J=8.1 Hz, 1H), 6.99 (d, J=8.1 Hz, 1H),
5.53-5.50 (m, 1H), 4.59-4.51 (m, 1H), 4.47 (s, 2H), 4.31 (s, 2H),
4.09 (s, 3H), 3.89 (s, 3H), 3.41-3.38 (m, 2H), 2.34-2.27 (m, 1H),
2.25-2.18 (m, 1H), 2.05-1.96 (m, 1H), 1.89-1.79 (m, 1H), 1.29 (d,
J=2.5 Hz, 3H), 1.27 (d, J=2.5 Hz, 3H). .sup.13C NMR (125 MHz,
CD.sub.3 OD) .delta. 168.1, 166.2, 163.4, 152.6, 147.8, 143.8,
134.2, 131.3, 130.2, 129.8, 129.5, 127.3, 126.8, 121.2, 106.7,
104.0, 55.4, 46.9, 43.6, 44.0, 42.0, 38.7, 31.1, 28.8, 23.7, 19.3,
19.3. HRMS m/z calculated for
C.sub.27H.sub.33ClN.sub.6O.sub.3(M+H.sup.+) 525.2374; found
525.2381.
(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(4-meth-
oxy-1-cyclopropyl-benzo[d]imidazol-2-yl)-L-ornithine) (8s)
##STR00116##
[0421] .sup.1H NMR (CD.sub.3 OD; 500 MHz): .delta. 8.03 (d, J=8.2
Hz, 1H), 7.64 (d, J=8.1 Hz, 1H), 7.58 (t, J=7.5 Hz, 1H), 7.44 (t,
J=8.2 Hz, 1H), 7.31 (d, J=8.1 Hz, 1H), 6.99 (d, J=8.2 Hz, 1H),
5.52-5.49 (m, 1H), 5.20 (d, J=45.5 Hz, 2H), 4.42 (s, 2H), 4.09 (s,
3H), 3.90 (s, 3H), 3.47-3.37 (m, 2H), 3.02-2.98 (m, 1H), 2.34-2.26
(m, 1H), 2.25-2.17 (m, 1H), 2.04-1.95 (m, 1H), 1.88-1.79 (m, 1H),
0.93-0.84 (m, 4H). .sup.13C NMR (125 MHz, CD.sub.3 OD) .delta.
170.3, 166.1, 152.6, 147.9, 143.7, 134.3, 131.5, 130.3, 129.8,
129.4, 127.3, 126.7, 121.4, 106.6, 104.0, 78.4, 76.9, 55.4, 50.0,
46.9, 41.4, 31.1, 28.8, 25.6, 23.8, 4.7, 4.5. HRMS m/z calculated
for C.sub.27H.sub.31FN.sub.6O.sub.3(M+H.sup.+) 507.2512; found
507.2516.
(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(4-meth-
oxy-1-cyclopropyl-benzo[d]imidazol-2-yl)-L-omithine) (8t)
##STR00117##
[0423] .sup.1H NMR (CD.sub.3 OD; 500 MHz): .delta. 8.02 (d, J=8.0
Hz, 1H), 7.63 (d, J=8.1 Hz, 1H), 7.58 (t, J=8.1 Hz, 1H), 7.43 (t,
J=8.2 Hz, 1H), 7.31 (d, J=8.2 Hz, 1H), 6.99 (d, J=8.2 Hz, 1H),
5.53-5.49 (m, 1H), 4.42 (s, 2H), 4.32 (s, 2H), 4.09 (s, 3H), 3.90
(s, 3H), 3.42-3.38 (m, 2H), 3.02-2.98 (m, 1H), 2.35-2.27 (m, 1H),
2.25-2.18 (m, 1H), 2.05-1.96 (m, 1H), 1.88-1.79 (m, 1H), 0.92-0.85
(m, 4H). .sup.13C NMR (125 MHz, CD.sub.3 OD) .delta. 170.3, 166.2,
163.5, 152.6, 147.8, 143.7, 134.2, 131.5, 130.2, 129.7, 129.3,
127.3, 126.6, 121.3, 106.6, 103.9, 55.3, 49.9, 46.9, 41.8, 38.8,
31.0, 28.7, 25.6, 23.6, 4.8, 4.5. HRMS m/z calculated for
C.sub.27H.sub.31ClN.sub.6O.sub.3(M+H.sup.+) 523.2216; found
523.2225.
(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(4-meth-
oxy-1-ethyl-benzo[d]imidazol-2-yl)-L-ornithine) (8u)
##STR00118##
[0425] .sup.1H NMR (CD.sub.3 OD; 500 MHz): .delta. 8.07 (dd, J=1.5
Hz, J=7.9 Hz, 1H), 7.69 (dd, J=1.5 Hz, J=7.4 Hz, 1H), 7.61 (t,
J=7.7 Hz, 1H), 7.44 (t, J=8.1 Hz, 1H), 7.34 (dd, J=1.0 Hz, J=8.2
Hz, 1H), 7.01 (d, J=8.3 Hz, 1H), 5.53-5.49 (m, 1H), 5.19 (d, J=45.2
Hz, 2H), 4.66-4.58 (m, 1H), 4.57-4.54 (m, 1H), 4.53 (s, 2H), 3.91
(s, 3H), 3.73-3.61 (m, 2H), 3.43-3.39 (m, 2H), 2.33-2.25 (m, 1H),
2.22-2.14 (m, 1H), 2.05-1.96 (m, 1H), 1.90-1.80 (m, 1H), 1.49 (t,
J=7.2 Hz, 3H), 1.25 (t, J=7.4 Hz, 3H). .sup.13C NMR (125 MHz,
CD.sub.3 OD) .delta. 168.6, 166.0, 152.2, 148.1, 143.7, 133.3,
131.4, 130.3, 129.8, 129.2, 127.2, 126.8, 121.8, 106.6, 104.1,
78.2, 76.9, 55.2, 49.2, 46.9, 41.5, 40.7, 37.7, 29.3, 23.9, 13.3,
12.2. HRMS m/z calculated for
C.sub.27H.sub.33FN.sub.6O.sub.3(M+H.sup.+) 509.2671; found
509.2671.
(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(4-meth-
oxy-1-ethyl-benzo[d]imidazol-2-yl)-L-ornithine)(8v)
##STR00119##
[0427] .sup.1H NMR (CD.sub.3 OD; 500 MHz): .delta. 8.06 (dd, J=1.4
Hz, J=8.1 Hz, 1H), 7.69 (dd, J=1.3 Hz, J=8.1 Hz, 1H), 7.60 (t,
J=7.7 Hz, 1H), 7.45 (t, J=8.4 Hz, 1H), 7.34 (dd, J=1.0 Hz, J=8.2
Hz, 1H), 7.00 (d, J=7.7 Hz, 1H), 5.53-5.50 (m, 1H), 4.65-4.59 (m,
1H), 4.58-4.53 (m, 1H), 4.52 (s, 2H), 4.30 (s, 2H), 3.90 (s, 3H),
3.71-3.62 (m, 2H), 3.41-3.37 (m, 2H), 2.33-2.25 (m, 1H), 2.23-2.15
(m, 1H), 2.06-1.97 (m, 1H), 1.91-1.81 (m, 1H), 1.50 (t, J=7.4 Hz,
3H), 1.25 (t, J=7.2 Hz, 3H). .sup.13C NMR (125 MHz, CD.sub.3 OD)
.delta. 168.5, 166.1, 163.3, 152.2, 148.0, 143.8, 133.2, 131.4,
130.4, 129.8, 129.3, 127.3, 126.8, 121.9, 106.5, 104.0, 55.4, 49.3,
46.9, 42.0, 40.7, 38.8, 37.7, 29.4, 23.8, 13.3, 12.1. HRMS m/z
calculated for C.sub.27H.sub.33ClN.sub.6O.sub.3(M+H.sup.+)
525.2375; found 525.2374.
(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(5-meth-
oxy-1H-benzo[d]imidazol-2-yl)-L-ornithine) (7w)
##STR00120##
[0429] .sup.1H NMR (500 MHz, CD.sub.3 OD) .delta. 8.03 (dd, J=2.5
Hz, J=8.7 Hz, 1H), 7.69 (d, J=8.1 Hz, 1H), 7.62 (t, J=8.2 Hz, 1H),
7.53 (d, J=8.8 Hz, 1H), 7.12 (s, 1H), 7.07 (dd, J=2.3 Hz, J=9.1 Hz,
1H), 5.49-5.46 (m, 1H), 5.18 (d, J=45.1 Hz, 2H), 4.53 (s, 2H), 3.78
(s, 3H), 3.69-3.65 (m, 2H), 3.42-3.38 (m, 1H), 3.27-3.22 (m, 1H),
1.95-1.79 (m, 2H), 1.24 (t, J=8.4 Hz, 3H). .sup.13C NMR (125 MHz,
CD.sub.3 OD) .delta. 168.6, 166.7, 161.7, 161.4, 161.2, 159.0,
153.4, 143.6, 132.3, 131.4, 130.3, 129.9, 129.1, 126.5, 125.3,
116.0, 114.3, 95.7, 78.2, 76.7, 55.0, 49.2, 44.2, 41.3, 38.8, 37.5,
29.6, 26.0, 23.7, 12.0. HRMS m/z calculated for
C.sub.25H.sub.29FN.sub.6O.sub.3(M+H.sup.+) 481.2358; found
481.2357.
(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(5-meth-
oxy-1H-benzo[d]imidazol-2-yl)-L-ornithine)(7x)
##STR00121##
[0431] .sup.1H NMR (500 MHz, CD.sub.3 OD) .delta. 8.02 (t, J=6.5
Hz, 1H), 7.68 (d, J=8.1 Hz, 1H), 7.61 (t, J=7.6 Hz, 1H), 7.52 (d,
J=8.5 Hz, 1H), 7.11 (s, 1H), 7.06 (dd, J=2.1 Hz, J=8.6 Hz, 1H),
5.50-5.47 (m, 1H), 4.52 (s, 3H), 4.29 (s, 2H), 3.77 (s, 3H),
3.69-3.63 (m, 2H), 3.40-3.36 (m, 1H), 3.28-3.22 (m, 1H), 2.27-2.19
(m, 2H), 1.95-1.80 (m, 2H), 1.24 (t, J=6.9 Hz, 3H). .sup.13C NMR
(125 MHz, CD.sub.3 OD) .delta. 168.4, 166.7, 163.4, 161.7, 161.2,
161.0, 159.1, 153.5, 143.6, 133.2, 131.3, 130.3, 129.9, 129.1,
126.4, 125.7, 125.2, 116.1, 114.3, 95.8, 55.1, 49.3, 44.1, 41.9,
38.8, 37.5, 29.7, 29.6, 25.9, 23.5, 22.4, 12.1. HRMS m/z calculated
for C.sub.25H.sub.29ClN.sub.6O.sub.3(M+H.sup.+) 497.2062; found
497.2063.
(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(5-meth-
oxy-1-ethyl-benzo[d]imidazol-2-yl)-L-ornithine) (7y)
##STR00122##
[0433] .sup.1H NMR (500 MHz, CD.sub.3 OD) .delta. 8.19 (d, J=8.2
Hz, 1H), 7.82 (t, J=6.6 Hz, 2H), 7.73 (t, J=7.8 Hz, 1H), 7.26-7.22
(m, 2H), 5.65-5.62 (m, 1H), 5.32 (d, J=45.6 Hz, 2H), 4.78-4.67 (m,
2H), 4.65 (s, 2H), 3.90 (s, 3H), 3.82-3.77 (m, 2H), 3.60-3.51 (m,
2H), 2.46-2.37 (m, 1H), 2.35-2.27 (m, 1H), 2.19-2.10 (m, 1H),
2.05-1.96 (m, 1H), 1.64 (t, J=8.0 Hz, 3H), 1.37 (t, J=7.4 Hz, 3H).
.sup.13C NMR (125 MHz, CD.sub.3 OD) .delta. 168.5, 166.2, 163.1,
163.0, 159.3, 152.0, 143.7, 131.9, 131.3, 130.3, 129.8, 129.3,
126.8, 125.8, 116.3, 113.2, 96.1, 78.3, 76.9, 55.1, 49.3, 46.7,
41.5, 40.6, 37.5, 29.3, 23.8, 13.4, 12.1. HRMS m/z calculated for
C.sub.27H.sub.33FN.sub.6O.sub.3(M+H.sup.+) 509.2671; found
509.2670.
(N1-(2-ethyl-3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(5-meth-
oxy-1-ethyl-benzo[d]imidazol-2-yl)-L-ornithine) (7z)
##STR00123##
[0435] .sup.1H NMR (500 MHz, CD.sub.3 OD) .delta. 8.06 (d, J=7.7
Hz, 1H), 7.70 (t, J=7.0 Hz, 2H), 7.61 (t, J=7.7 Hz, 1H), 7.14-7.09
(m, 2H), 5.53-5.49 (m, 1H), 4.65-4.54 (m, 2H), 4.53 (s, 2H), 4.30
(s, 2H), 3.77 (s, 3H), 3.69-3.64 (m, 2H), 3.42-3.38 (m, 2H),
2.32-2.24 (m, 1H), 2.22-2.15 (m, 1H), 2.06-1.98 (m, 1H), 1.93-1.84
(m, 1H), 1.51 (t, J=7.0 Hz, 3H), 1.24 (t, J=7.0 Hz, 3H). .sup.13C
NMR (125 MHz, CD.sub.3 OD) .delta. 168.5, 166.2, 163.4, 161.0,
159.5, 151.9, 143.7, 131.9, 131.4, 130.3, 129.8, 129.3, 126.8,
125.9, 116.3, 113.3, 96.1, 55.1, 49.3, 46.7, 41.9, 40.7, 38.7,
37.5, 29.3, 23.6, 13.4, 12.0. HRMS m/z calculated for
C.sub.27H.sub.33ClN.sub.6O.sub.3(M+H.sup.+) 525.2375; found
525.2378.
(N1-(2-methyl-3-oxoisoindoline)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(4-met-
hoxy-1-ethyl-benzo[d]imidazol-2-yl)-L-ornithine) (8a')
##STR00124##
[0437] .sup.1H NMR (500 MHz, CD.sub.3 OD) .delta. 8.05 (d, J=8.1
Hz, 1H), 7.67 (d, J=8.1 Hz, 1H), 7.59 (t, J=7.4 Hz, 1H), 7.45 (t,
J=9.1 Hz, 1H), 7.32 (d, J=9.1 Hz, 1H), 7.01 (d, J=8.1 Hz, 1H),
5.52-5.49 (m, 1H), 5.19 (d, J=45.6 Hz, 2H), 4.50 (s, 2H), 4.09 (s,
3H), 3.90 (s, 3H), 3.44-3.39 (m, 2H), 3.18 (s, 3H), 2.34-2.26 (m,
1H), 2.23-2.16 (m, 1H), 2.05-1.96 (m, 1H), 1.88-1.80 (m, 1H).
.sup.13C NMR (125 MHz, CD.sub.3 OD) .delta. 168.9, 166.1, 163.2,
163.0, 161.1, 160.8, 152.5, 147.8, 143.6, 134.2, 131.4, 130.3,
129.6, 129.1, 127.3, 126.6, 121.3, 106.7, 104.0, 78.3, 76.9, 55.3,
51.8, 46.8, 41.4, 31.0, 29.0, 28.7, 23.8. HRMS m/z calculated for
C.sub.25H.sub.29FN.sub.6O.sub.3(M+H.sup.+) 481.2358; found
481.2359.
(N1-(2-methyl-3-oxoisoindoline)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(4-met-
hoxy-1-ethyl-benzo[d]imidazol-2-yl)-L-ornithine) (8b')
##STR00125##
[0439] .sup.1H NMR (500 MHz, CD.sub.3 OD) .delta. 8.05 (d, J=7.6
Hz, 1H), 7.67 (d, J=7.7 Hz, 1H), 7.59 (t, J=7.4 Hz, 1H), 7.44 (t,
J=8.3 Hz, 1H), 7.32 (d, J=8.3 Hz, 1H), 7.01 (d, J=8.8 Hz, 1H),
5.52-5.49 (m, 1H), 4.50 (s, 2H), 4.31 (s, 2H), 4.09 (s, 3H), 3.90
(s, 3H), 3.41-3.38 (m, 2H), 3.18 (s, 3H), 2.34-2.26 (m, 1H),
2.25-2.17 (m, 1H), 2.05-1.96 (m, 1H), 1.89-1.80 (m, 1H). .sup.13C
NMR (125 MHz, CD.sub.3 OD) .delta. 168.9, 166.1, 163.4, 161.1,
160.7, 152.6, 147.8, 143.6, 134.3, 131.4, 130.3, 129.7, 129.2,
127.3, 126.6, 121.3, 106.7, 103.9, 55.3, 51.8, 46.8, 41.9, 38.7,
31.1, 29.0, 28.8, 23.7. HRMS m/z calculated for
C.sub.25H.sub.29ClN.sub.6O.sub.3(M+H.sup.+) 497.2062; found
497.2062.
6. General Procedure for Synthesis of Benzimidazole
haloacetamidines 9af..sup.a
##STR00126##
[0441] To a stirred solution of 4b-d (1.0 eq) in DMF was added HOBt
(2.0 eq), HBTU (2.0 eq), and DIPEA (3.0 eq) followed by
2-(tert-butoxycarbonyl)benzoic acid (1.0 eq) and allowed to stir at
rt for 12 h. The reaction mixture was then diluted with water. The
product was filtered, washed with water, dried under vacuum, and
obtained in 62-71% yield. This product was then treated with 1 M
HCl in EtOAc to remove the Boc group giving the
CO.sub.2.sup.tBu-Bz-Orn-benzimidazole intermediate. The solvent was
then evaporated to dryness and the crude material was dried in
vacuo. To a stirred solution of the corresponding
CO.sub.2.sup.tBu-Bz-Orn-benzimidazole intermediate in dry MeOH was
added TEA (4.0 eq) followed by ethyl haloacetimidate HCl (2.0 eq).
The reaction was stirred under N.sub.2 at rt for 3 h. Solvents were
then evaporated under reduced pressure and the crude product was
purified by reverse phase HPLC using MeCN:H.sub.2O (0.5% TFA) as an
eluent to give compounds 9a-f in 51-67% yield.
(N1-(2-tert-butylcarboxy)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-methyl-be-
nzo[d]imidazol-2-yl)-L-ornithine (9a)
##STR00127##
[0443] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 7.85-7.81 (m,
2H), 7.77 (dd, J=1.7 Hz, J=7.2 Hz, 1H), 7.63-7.51 (m, 4H), 7.48
(dd, J=1.3 Hz, J=7.6 Hz, 1H), 5.68-5.65 (m, 1H), 5.27 (d, J=45.3
Hz, 2H), 4.19 (s, 3H), 3.54-3.46 (m, 2H), 2.42-2.23 (m, 2H),
2.09-1.98 (m, 1H), 1.95-1.85 (m, 1H), 1.32 (s, 9H). .sup.13C NMR
(100 MHz, CD.sub.3 OD) .delta. 170.9, 165.0, 151.4, 150.0, 144.4,
143.8, 131.5, 130.4, 129.8, 129.5, 125.4, 125.2, 111.7, 100.2,
82.7, 81.0, 78.4, 76.1, 54.0, 53.2, 45.1, 40.6, 30.6, 28.4, 26.4,
23.2. HRMS m/z calculated for
C.sub.26H.sub.32FN.sub.5O.sub.3(M+H.sup.+) 482.2562; found
482.2561.
(N1-(2-tert-butylcarboxy)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-methyl-be-
nzo[d]imidazol-2-yl)-L-ornithine) (9b)
##STR00128##
[0445] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 7.89-7.78 (m,
3H), 7.63-7.46 (m, 5H), 5.69-5.64 (m, 1H), 4.37 (s, 2H), 4.21 (s,
3H), 3.50-3.44 (m, 2H), 2.43-2.23 (m, 2H), 2.11-1.99 (m, 1H),
1.96-1.85 (m, 1H), 1.32 (s, 9H). .sup.13C NMR (100 MHz, CD.sub.3
OD) .delta. 165.3, 163.6, 152.3, 136.4, 131.5, 131.3, 130.4, 129.7,
129.3, 126.9, 125.3, 114.7, 111.5, 94.6, 81.2, 72.6, 72.0, 66.5,
50.0, 44.8, 42.0, 38.5, 30.2, 28.4, 26.3, 23.3. HRMS m/z calculated
for C.sub.26H.sub.32ClN.sub.5O.sub.3(M+H.sup.+) 498.2266; found
498.2268.
(N1-(2-tert-butylcarboxy)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-ethyl-ben-
zo[d]imidazol-2-yl)-L-ornithine) (9c)
##STR00129##
[0447] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 7.97 (dd, J=2.3
Hz, J=6.8 Hz, 1H), 7.85-7.81 (m, 2H), 7.68-7.58 (m, 3H), 7.56-7.49
(m, 2H), 5.70-5.65 (m, 1H), 5.26 (d, J=45.3 Hz, 2H), 4.89-4.81 (m,
1H), 4.76-4.68 (m, 1H), 3.50-3.45 (m, 2H), 2.42-2.20 (m, 2H),
2.12-2.00 (m, 1H), 1.94-1.81 (m, 1H), 1.64 (t, J=7.5 Hz, 3H), 1.32
(s, 9H). .sup.13C NMR (100 MHz, CD.sub.3 OD) .delta. 171.6, 165.2,
163.3, 163.2, 162.4, 161.0, 151.7, 136.7, 131.8, 130.4, 129.7,
129.5, 127.1, 126.6, 126.3, 114.3, 112.7, 81.6, 78.6, 76.7, 45.6,
41.8, 40.4, 28.8, 26.9, 23.8, 13.4. HRMS m/z calculated for
C.sub.27H.sub.34FN.sub.5O.sub.3(M+H.sup.+) 496.2718; found
496.2718.
(N1-(2-tert-butylcarboxy)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-ethyl-ben-
zo[d]imidazol-2-yl)-L-ornithine) (9d)
##STR00130##
[0449] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 7.98 (dd, J=2.1
Hz, J=7.2 Hz, 1H), 7.86-7.81 (m, 2H), 7.68-7.62 (m, 2H), 7.60 (dd,
J=1.3 Hz, J=7.5 Hz, 1H), 7.56-7.49 (m, 2H), 5.70-5.65 (m, 1H),
4.90-4.82 (m, 1H), 4.77-4.68 (m, 1H), 4.38 (s, 2H), 3.48-3.43 (m,
2H), 2.43-2.21 (m, 2H), 2.15-2.02 (m, 1H), 1.95-1.83 (m, 1H), 1.64
(t, J=7.3 Hz, 3H), 1.32 (s, 9H). .sup.13C NMR (100 MHz, CD.sub.3
OD) .delta. 171.6, 165.3, 163.3, 160.4, 160.1, 159.8, 151.4, 136.1,
132.2, 130.5, 130.2, 128.4, 127.3, 126.3, 108.9, 88.7, 81.4, 50.4,
45.9, 44.5, 38.2, 36.4, 31.7, 28.4, 27.0, 25.3, 13.8. HRMS m/z
calculated for C.sub.27H.sub.34ClN.sub.5O.sub.3(M+H.sup.+)
512.2423; found 512.2422.
(N1-(2-tert-butylcarboxy)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-isopropyl-
-benzo[d]imidazol-2-yl)-L-omithine) (9e)
##STR00131##
[0451] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.12 (m, 1H),
7.86-7.81 (m, 2H), 7.64-7.58 (m, 3H), 7.55 (dd, J=1.4 Hz, J=7.7 Hz,
1H), 7.52-7.48 (m, 1H), 5.74-5.69 (m, 1H), 5.47-5.39 (m, 1H), 5.26
(d, J=45.4 Hz, 2H), 3.52-3.44 (m, 2H), 2.42-2.20 (m, 2H), 2.11-1.99
(m, 1H), 1.94-1.86 (m, 1H), 1.85 (d, J=3.3 Hz, 3H), 1.83 (d, J=3.4
Hz, 3H), 1.34 (s, 9H). .sup.13C NMR (100 MHz, CD.sub.3 OD) .delta.
171.5, 165.3, 163.0, 161.8, 151.2, 136.3, 132.3, 131.7, 130.6,
130.2, 129.8, 129.4, 127.4, 126.0, 125.4, 114.9, 114.7, 81.4, 78.5,
76.1, 51.0, 45.1, 41.4, 28.7, 26.3, 23.7, 19.9, 19.8. HRMS m/z
calculated for C.sub.28H.sub.36FN.sub.5O.sub.3(M+H.sup.+) 510.2875;
found 510.2878.
(N1-(2-tert-butylcarboxy)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-isopropyl-
-benzo[d]imidazol-2-yl)-L-omithine) (9f)
##STR00132##
[0453] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.12-8.07 (m,
1H), 7.85-7.81 (m, 2H), 7.63-7.57 (m, 3H), 7.55 (dd, J=1.3 Hz,
J=7.8 Hz, 1H), 7.52-7.48 (m, 1H), 5.73-5.68 (m, 1H), 5.43-5.46 (m,
1H), 4.37 (s, 2H), 3.47-3.42 (m, 2H), 2.41-2.21 (m, 2H), 2.10-1.98
(m, 1H), 1.93-1.85 (m, 1H), 1.84 (d, J=3.1 Hz, 3H), 1.82 (d, J=3.2
Hz, 3H), 1.36 (9H). .sup.13C NMR (100 MHz, CD.sub.3 OD) .delta.
170.9, 168.8, 165.0, 163.1, 151.0, 136.4, 131.4, 130.5, 130.3,
129.7, 129.3, 127.1, 125.6, 125.2, 115.1, 114.4, 81.5, 77.0, 76.1,
50.9, 45.5, 41.8, 38.7, 28.9, 26.7, 23.4, 19.9, 19.8. HRMS m/z
calculated for C.sub.28H.sub.36ClN.sub.5O.sub.3(M+H.sup.+)
526.2579; found 526.2577.
6. General Procedure for Synthesis of Benzimidazole
haloacetamidines 10a-h and 11a-h
##STR00133##
[0455] To a stirred solution of 4a-d (1.0 eq) in THF was added
5-phenylisobenzofuran-1,3-dione (1.0 eq) and allowed to stir at rt
under N.sub.2 for 18 h. Solvents were evaporated and the crude
product was purified by reverse phase HPLC using MeCN:H.sub.2O
(0.5% TFA) as the eluent to give the product in 78-86% yield. This
product was then treated with TFA to remove the Boc group giving
the 4-Ph-2-CO.sub.2H-Bz-Orn-benzimidazole and
3-Ph-2-CO.sub.2H-Bz-Orn intermediates as a .about.50:50 mixture.
The solvent was then evaporated to dryness and the crude material
was dried in vacuo. To a stirred solution of the corresponding
4-Ph-2-CO.sub.2H-Bz-Orn-benzimidazole and 3-Ph-2-CO.sub.2H-Bz-Orn
mixture in dry MeOH was added TEA (4.0 eq) followed by ethyl
haloacetimidate HCl (2.0 eq). The reaction was stirred under
N.sub.2 at rt for 3 h. Solvents were then evaporated under reduced
pressure and the crude product was purified by reverse phase HPLC
using MeCN:H.sub.2O (0.5% TFA) as an eluent to give compounds 10a-h
and 11a-h in 55-69% yield.
(N1-[1,4'-Phenyl](2-carboxyl)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1H-benz-
o[d]imidazol-2-yl)-L-ornithine) (10a) and
(N1-[1,3'-Phenyl](2-carboxyl)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1H-ben-
zo[d]imidazol-2-yl)-L-omithine) (11a)
##STR00134##
[0457] Compounds 10a and 11a were isolated as a .about.40:60
mixture, respectively.
[0458] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.27 (d, J=1.6
Hz, 0.4H), 8.14 (d, J=8.2 Hz, 0.6H), 7.93 (dd, J=1.4 Hz, J=7.8 Hz,
0.4H), 7.86 (dd, J=1.5 Hz, J=7.9 Hz, 0.6H), 7.81-7.63 (m, 5H),
7.54-7.48 (m, 4H), 7.43 (t, J=7.4 Hz, 1H), 5.56-5.52 (m, 1H), 5.29
(d, J=45.3 Hz, 0.4H), 5.26 (d, J=45.4 Hz, 0.6H), 3.52-3.45 (m, 2H),
2.38-2.21 (m, 2H), 2.07-1.87 (m, 2H). .sup.13C NMR (125 MHz,
CD.sub.3 OD) .delta. 171.8, 168.1, 167.9, 162.1, 154.7, 154.4,
154.2, 153.5, 145.3, 142.8, 138.9, 138.8, 138.4, 133.6, 131.0,
130.2, 129.8, 128.8, 128.5, 128.4, 128.1, 127.8, 126.9, 126.6,
126.0, 125.6, 124.8, 114.0, 99.9, 78.3, 76.9, 67.3, 41.5, 28.9,
25.1, 23.5. HRMS m/z calculated for
C.sub.27H.sub.26FN.sub.5O.sub.3(M+H.sup.+) 488.2092; found
488.2091.
(N1-[1,4'-Phenyl](2-carboxyl)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1H-benz-
o[d]imidazol-2-yl)-L-ornithine) (10b) and
(N1-[1,3'-Phenyl](2-carboxyl)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1H-ben-
zo[d]imidazol-2-yl)-L-omithine) (11b)
##STR00135##
[0459] Compounds 10b and 11b were isolated as a .about.40:60
mixture, respectively.
[0460] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.26 (d, J=1.9
Hz, 0.4H), 8.13 (d, J=8.2 Hz, 0.6H), 7.93 (dd, J=2.1 Hz, J=8.1 Hz,
0.4H), 7.85 (dd, J=1.8 Hz, J=8.2 Hz, 0.6H), 7.81-7.63 (m, 5H),
7.55-7.48 (m, 4H), 7.44 (J=7.06 Hz, 1H), 5.56-5.52 (m, 1H), 4.39
(s, 0.8H), 4.37 (s, 1.2H), 3.49-3.43 (m, 2H), 2.36-2.23 (m, 2H),
2.07-1.87 (m, 2H). .sup.13C NMR (125 MHz, CD.sub.3 OD) .delta.
171.9, 171.8, 168.2, 167.8, 167.4, 163.2, 161.6, 161.1, 153.2,
145.1, 138.9, 138.5, 138.0, 135.9, 132.3, 131.0, 130.3, 129.8,
128.8, 128.3, 127.8, 127.3, 126.8, 126.6, 125.8, 125.3, 113.7,
41.9, 38.5, 28.9, 23.4, 20.2. HRMS m/z calculated for
C.sub.27H.sub.26ClN.sub.5O.sub.3(M+H.sup.+) 504.1797; found
504.1798.
(N1-[1,4'-Phenyl](2-carboxyl)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-methy-
l-benzo[d]imidazol-2-yl)-L-ornithine) (10c) and
(N1-[1,3'-Phenyl](2-carboxyl)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-meth-
yl-benzo[d]imidazol-2-yl)-L-ornithine) (11c)
##STR00136##
[0461] Compounds 10c and 11c were isolated as a .about.20:80
mixture, respectively.
[0462] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.21 (d, J=2.1
Hz, 0.2H), 8.07 (d, J=8.1 Hz, 0.8H), 7.92-7.78 (m, 3H), 7.72-7.57
(m, 5H), 7.50-7.38 (m, 2H), 5.71-5.67 (m, 1H), 5.28 (d, J=45.3 Hz,
0.2H), 5.26 (d, J=45.4 Hz, 0.8H), 4.24 (s, 3H), 3.55-3.43 (m, 2H),
2.43-2.33 (m, 1H), 2.31-2.21 (m, 1H), 2.13-2.01 (m, 1H), 1.96-1.84
(m, 1H). .sup.13C NMR (100 MHz, CD.sub.3 OD) .delta. 171.6, 166.8,
163.1, 151.7, 145.1, 138.6, 137.6, 132.5, 130.8, 128.8, 128.3,
127.8, 127.4, 126.8, 126.6, 126.3, 125.9, 125.7, 114.9, 114.3,
112.2, 78.3, 76.6, 45.5, 41.3, 30.8, 28.3, 25.3. HRMS m/z
calculated for C.sub.28H.sub.28FN.sub.5O.sub.3(M+H.sup.+) 502.2249;
found 502.2255.
(N1-[1,4'-Phenyl](2-carboxyl)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-methy-
l-benzo[d]imidazol-2-yl)-L-ornithine) (10d) and
(N1-[1,3'-Phenyl](2-carboxyl)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-meth-
yl-benzo[d]imidazol-2-yl)-L-ornithine) (11d)
##STR00137##
[0463] Compounds 10d and 11d were isolated as a .about.20:80
mixture, respectively.
[0464] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.20 (d, J=2.0
Hz, 0.2H), 8.08 (d, J=8.4 Hz, 0.8H), 7.90-7.78 (m, 3H), 7.72-7.62
(m, 2H), 7.60-7.55 (m, 3H), 7.51-7.38 (m, 2H), 5.70-5.66 (m, 1H),
4.39 (s, 1.6H), 4.38 (s, 0.4H), 4.22 (s, 2.4H), 4.21 (s, 0.6H),
3.52-3.43 (m, 2H), 2.44-2.23 (m, 1H), 2.11-1.85 (m, 1H). .sup.13C
NMR (100 MHz, CD.sub.3 OD) .delta. 171.9, 171.7, 168.1, 167.8,
163.3, 161.7, 161.3, 153.3, 145.2, 142.8, 138.9, 138.7, 138.3,
136.0, 132.2, 131.1, 130.2, 129.8, 128.7, 128.4, 128.3, 128.2,
128.0, 127.9, 127.4, 126.8, 126.7, 125.9, 125.5, 113.8, 41.9, 38.7,
28.9, 23.5. HRMS m/z calculated for
C.sub.28H.sub.28ClN.sub.5O.sub.3(M+H.sup.+) 518.1953; found
518.1957.
(N1-[1,4'-Phenyl](2-carboxyl)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-ethyl-
-benzo[d]imidazol-2-yl)-L-ornithine) (10e) and
(N1-[1,3'-Phenyl](2-carboxyl)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-ethy-
l-benzo[d]imidazol-2-yl)-L-ornithine) (11e)
##STR00138##
[0465] Compounds 10e and 11e were isolated as a .about.40:60
mixture, respectively.
[0466] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.20 (d, 0.4H),
8.07 (d, J=9.6 Hz, 0.6H), 7.94-7.80 (m, 3H) 7.73-7.57 (m, 5H), 7.48
(t, J=8.0 Hz, 2H), 7.43-7.37 (m, 1H), 5.69-5.65 (m, 1H), 5.27 (d,
J=45.2 Hz, 0.8H), 5.26 (d, J=45.4 Hz, 1.2H), 4.85-4.77 (m, 1H),
4.74-4.65 (m, 1H), 3.53-3.41 (m, 2H), 2.42-2.30 (m, 1H), 2.29-2.19
(m, 1H), 2.13-2.01 (m, 1H), 1.96-1.84 (m, 1H), 1.64 (t, J=8.0 Hz,
3H). .sup.13C NMR (100 MHz, CD.sub.3 OD) .delta. 171.7, 171.5,
167.3, 167.0, 163.0, 162.8, 161.4, 161.2, 151.6, 145.0, 142.9,
138.9, 138.8, 137.8, 135.6, 131.6, 131.5, 130.9, 130.0, 129.8,
128.7, 128.3, 128.3, 128.1, 128.0, 127.9, 127.6, 126.8, 126.6,
126.3, 126.0, 125.7, 118.0, 115.1, 114.6, 112.4, 78.4, 76.7, 45.6,
41.4, 40.6, 29.0, 23.7, 13.6. HRMS m/z calculated for
C.sub.29H.sub.30FN.sub.5O.sub.3(M+H.sup.+) 516.2405; found
516.2405.
(N1-[1,4'-Phenyl](2-carboxyl)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-ethyl-
-benzo[d]imidazol-2-yl)-L-ornithine) (10f) and
(N1-[1,3'-Phenyl](2-carboxyl)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-ethy-
l-benzo[d]imidazol-2-yl)-L-ornithine) (11f)
##STR00139##
[0467] Compounds 10f and 11f were isolated as a .about.40:60
mixture, respectively.
[0468] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.20 (d, J=2.3
Hz, 0.4H), 8.07 (dd, J=1.9 Hz, J=9.6 Hz, 0.6H), 7.94-7.80 (m, 3H),
7.73-7.57 (m, 5H), 7.48 (t, J=7.4 Hz, 2H), 7.43-7.38 (m, 1H),
5.70-5.65 (m, 1H), 4.86-4.76 (m, 1H), 4.73-4.65 (m, 1H), 4.39 (s,
0.8H), 4.38 (s, 1.2H),4.37 (s, 0.4H), 4.36 (s, 0.6H), 3.50-3.40 (m,
2H), 2.41-2.31 (m, 1H), 2.29-2.20 (m, 1H), 2.13-2.02 (m, 1H),
1.96-1.85 (m, 1H), 1.64 (t, J=7.5 Hz, 3H). .sup.13C NMR (100 MHz,
CD.sub.3 OD) .delta. 171.7, 171.5, 167.4, 167.1, 163.3, 161.5,
161.2, 151.7, 145.0, 142.9, 138.9, 138.8, 137.9, 135.7, 131.7,
130.9, 130.1, 129.9, 128.8, 128.4, 128.3, 128.0, 128.0, 127.9,
127.6, 126.8, 126.6, 126.3, 126.0, 125.8, 118.0, 115.1, 114.6,
112.4, 45.6, 45.5, 41.9, 40.6, 38.7, 29.0, 28.9, 23.6, 13.6. HRMS
m/z calculated for C.sub.29H.sub.30ClN.sub.5O.sub.3(M+H.sup.+)
532.2110; found 532.2112.
(N1-[1,4'-Phenyl](2-carboxyl)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-isopr-
opyl-benzo[d]imidazol-2-yl)-L-ornithine) (10g) and
(N1-[1,3'-Phenyl](2-carboxyl)benzoyl-N5-(2-fluoro-1-iminoethyl)-1-(1-isop-
ropyl-benzo[d]imidazol-2-yl)-L-ornithine) (11g)
##STR00140##
[0469] Compounds 10g and 11g were isolated as a .about.40:60
mixture, respectively.
[0470] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.20 (d, J=2.2
Hz, 0.4H), 8.11-8.04 (m, 1.6H), 7.90-7.79 (m, 2H), 7.72-7.63 (m,
3H), 7.60-7.54 (m, 2H), 7.50-7.45 (m, 2H), 7.44-7.37 (m, 1H),
5.72-5.66 (m, 1H), 5.40-5.33 (m, 1H), 5.27 (d, J=45.3 Hz, 0.4H),
5.21 (d, J=45.2 Hz, 0.6H), 3.53-3.43 (m, 2H), 2.40-2.29 (m, 1H),
2.28-2.19 (m, 1H), 2.10-1.98 (m, 1H), 1.94-1.85 (m, 1H), 1.85 (d,
J=7.3 Hz, 6H). .sup.13C NMR (100 MHz, CD.sub.3 OD) .delta. 171.6,
171.3, 167.4, 167.2, 163.1, 161.4, 161.0, 161.4, 151.4, 145.0,
143.0, 138.9, 138.8, 137.8, 135.6, 130.9, 130.4, 130.1, 130.0,
128.8, 128.4, 128.3, 128.0, 128.0, 127.6, 126.8, 126.6, 125.8,
125.8, 125.4, 115.1, 114.6, 78.4, 76.6, 51.1, 45.9, 45.8, 41.4,
29.2, 29.1, 23.7, 19.8, 19.8. HRMS m/z calculated for
C.sub.30H.sub.32FN.sub.5O.sub.3(M+H.sup.+) 530.2562; found
530.2562.
(N1-[1,4'-Phenyl](2-carboxyl)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-isopr-
opyl-benzo[d]imidazol-2-yl)-L-ornithine) (10h) and
(N1-[1,3'-Phenyl](2-carboxyl)benzoyl-N5-(2-chloro-1-iminoethyl)-1-(1-isop-
ropyl-benzo[d]imidazol-2-yl)-L-ornithine) (11h)
##STR00141##
[0471] Compounds 10h and 11h were isolated as a .about.40:60
mixture, respectively.
[0472] .sup.1H NMR (CD.sub.3 OD; 400 MHz): .delta. 8.20 (d, J=2.2
Hz, 0.4H), 8.10-8.06 (m, 1.6H), 7.90-7.80 (m, 2H), 7.72-7.64 (m,
3H), 7.60-7.55 (m, 2H), 7.50-7.45 (m, 2H), 7.43-7.38 (m, 1H),
5.72-5.68 (m, 1H), 5.41-5.31 (m, 1H), 4.38 (s, 0.8H), 4.37 (s,
1.2H), 3.49-3.41 (m, 2H), 2.41-2.31 (m, 1H), 2.29-2.20 (m, 1H),
2.11-1.98 (m, 1H), 1.94-1.86 (m, 1H), 1.84-1.82 (d, J=7.1 Hz, 6H).
.sup.13C NMR (100 MHz, CD.sub.3 OD) .delta. 171.6, 167.5, 167.1,
164.8, 163.3, 161.5, 161.0, 151.4, 146.7, 145.0, 142.9, 141.8,
138.8, 137.9, 137.3, 135.6, 130.9, 130.5, 130.2, 130.0, 128.7,
128.4, 128.2, 128.0, 127.9, 127.6, 126.8, 126.6, 125.7, 125.4,
115.1, 114.6, 110.0, 51.0, 45.9, 45.8, 41.9, 38.7, 29.1, 23.6,
19.9, 19.8. HRMS m/z calculated for C.sub.30H.sub.32FN.sub.5O.sub.3
(M+H.sup.+) 546.2266; found 546.2270.
[0473] Applicant's disclosure is described herein in preferred
embodiments with reference to the Figures, in which like numbers
represent the same or similar elements. Reference throughout this
specification to "one embodiment," "an embodiment," or similar
language means that a particular feature, structure, or
characteristic described in connection with the embodiment is
included in at least one embodiment of the present invention. Thus,
appearances of the phrases "in one embodiment," "in an embodiment,"
and similar language throughout this specification may, but do not
necessarily, all refer to the same embodiment.
[0474] The described features, structures, or characteristics of
Applicant's disclosure may be combined in any suitable manner in
one or more embodiments. In the description herein, numerous
specific details are recited to provide a thorough understanding of
embodiments of the invention. One skilled in the relevant art will
recognize, however, that Applicant's composition and/or method may
be practiced without one or more of the specific details, or with
other methods, components, materials, and so forth. In other
instances, well-known structures, materials, or operations are not
shown or described in detail to avoid obscuring aspects of the
disclosure.
[0475] In this specification and the appended claims, the singular
forms "a," "an," and "the" include plural reference, unless the
context clearly dictates otherwise.
[0476] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art. Although any methods and materials
similar or equivalent to those described herein can also be used in
the practice or testing of the present disclosure, the preferred
methods and materials are now described. Methods recited herein may
be carried out in any order that is logically possible, in addition
to a particular order disclosed.
INCORPORATION BY REFERENCE
[0477] References and citations to other documents, such as
patents, patent applications, patent publications, journals, books,
papers, web contents, have been made in this disclosure. All such
documents are hereby incorporated herein by reference in their
entirety for all purposes. Any material, or portion thereof, that
is said to be incorporated by reference herein, but which conflicts
with existing definitions, statements, or other disclosure material
explicitly set forth herein is only incorporated to the extent that
no conflict arises between that incorporated material and the
present disclosure material. In the event of a conflict, the
conflict is to be resolved in favor of the present disclosure as
the preferred disclosure.
EQUIVALENTS
[0478] The representative examples are intended to help illustrate
the invention, and are not intended to, nor should they be
construed to, limit the scope of the invention. Indeed, various
modifications of the invention and many further embodiments
thereof, in addition to those shown and described herein, will
become apparent to those skilled in the art from the full contents
of this document, including the examples and the references to the
scientific and patent literature included herein. The examples
contain important additional information, exemplification and
guidance that can be adapted to the practice of this invention in
its various embodiments and equivalents thereof.
* * * * *