U.S. patent application number 14/425909 was filed with the patent office on 2015-10-15 for novel compound with effects of thrombolysis, free radical scavenging and thrombus-targeting as well as preparation method and use thereof.
The applicant listed for this patent is Shanghai Lumosa Therapeutics, Co., Ltd.. Invention is credited to Xueyun Jiang, Shiqi Peng, Ming Zhao.
Application Number | 20150290339 14/425909 |
Document ID | / |
Family ID | 50236496 |
Filed Date | 2015-10-15 |
United States Patent
Application |
20150290339 |
Kind Code |
A1 |
Peng; Shiqi ; et
al. |
October 15, 2015 |
NOVEL COMPOUND WITH EFFECTS OF THROMBOLYSIS, FREE RADICAL
SCAVENGING AND THROMBUS-TARGETING AS WELL AS PREPARATION METHOD AND
USE THEREOF
Abstract
The present invention discloses a novel compound with effects of
thrombolysis, free radical scavenging and thrombus-targeting, as
well as a preparation method and use thereof. The compound is a
ternary conjugate formed by conjugating a thrombolytic peptide, a
free radical scavenger and a thrombus-targeting/antithrombotic
peptide together via a linking arm. The present invention also
discloses a pharmaceutical composition containing the compounds,
wherein the compounds form a nanospherical structure.
Inventors: |
Peng; Shiqi; (Beijing,
CN) ; Zhao; Ming; (Beijing, CN) ; Jiang;
Xueyun; (Beijing, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Shanghai Lumosa Therapeutics, Co., Ltd. |
Shanghai Pilot Free Trade Zone |
|
CN |
|
|
Family ID: |
50236496 |
Appl. No.: |
14/425909 |
Filed: |
March 15, 2013 |
PCT Filed: |
March 15, 2013 |
PCT NO: |
PCT/CN2013/072731 |
371 Date: |
April 17, 2015 |
Current U.S.
Class: |
424/489 ;
514/14.9; 530/328 |
Current CPC
Class: |
A61P 7/12 20180101; A61P
9/00 20180101; A61P 29/00 20180101; A61K 38/07 20130101; A61P 7/02
20180101; A61K 38/00 20130101; A61P 3/10 20180101; C07K 7/06
20130101; A61K 38/06 20130101; A61K 47/542 20170801; A61K 47/6929
20170801; A61K 31/4164 20130101; C07K 5/0815 20130101; A61P 9/10
20180101; A61P 25/28 20180101; A61P 25/18 20180101; A61K 38/08
20130101; A61P 35/00 20180101; A61P 19/02 20180101; A61P 25/00
20180101; C07K 5/0821 20130101; A61P 39/06 20180101; C07K 5/0806
20130101; A61K 47/64 20170801; A61P 35/02 20180101; A61P 17/00
20180101 |
International
Class: |
A61K 47/48 20060101
A61K047/48; A61K 38/06 20060101 A61K038/06; A61K 38/08 20060101
A61K038/08; A61K 38/07 20060101 A61K038/07; C07K 7/06 20060101
C07K007/06; A61K 31/4164 20060101 A61K031/4164 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 5, 2012 |
CN |
201210323848.9 |
Sep 5, 2012 |
CN |
201210323849.3 |
Sep 5, 2012 |
CN |
201210323850.6 |
Sep 5, 2012 |
CN |
201210323951.3 |
Mar 5, 2013 |
CN |
201310068532.4 |
Claims
1. A ternary conjugate consisting of an imidazoline having NO free
radical scavenging activity, a peptide having thrombolytic
activity, and a thrombus-targeting peptide, which are linked
together via a suitable linking arm.
2. The ternary conjugate according to claim 1, represented by
formula I: ##STR00012## wherein NN represents an imidazoline having
NO free radical scavenging activity; AA.sub.1 represents a linking
arm having at least three groups for linking; AA.sub.2 represents a
peptide having thrombolytic activity; and AA.sub.3 represents a
thrombus-targeting peptide.
3. The ternary conjugate according to claim 1, wherein the
imidazoline having NO free radical scavenging activity is
1,3-dioxo-2-[(4-oxyacetoxy)phenyl]-4,4,5,5-tetramethylimidazoline.
4. The ternary conjugate according to claim 1, wherein the groups
for linking are selected from the group consisting of a carboxyl
group and an amino group.
5. The ternary conjugate according to claim 4, wherein the linking
arm is a natural amino acid, particularly L-Lys, L-Asp or
L-Glu.
6. The ternary conjugate according to claim 1, wherein the peptide
having thrombolytic activity is an oligopeptide comprising a PAK
(Pro-Ala-Lys) sequence, an AKP (Ala-Lys-Pro) sequence or a KAP
(Lys-Ala-Pro) sequence, or a peptide having repeating units of the
PAK sequence, the AKP sequence or the KAP sequence.
7. The ternary conjugate according to claim 1, wherein the
thrombus-targeting peptide is an oligopeptide comprising an RGD
(Arg-Gly-Asp) sequence.
8. The ternary conjugate according to claim 1, wherein the
thrombus-targeting peptide is a polypeptide obtained from
conjugating modification of an RGD (Arg-Gly-Asp) peptide with a
YIGS (Tyr-Ile-Gly-Ser) peptide.
9. The ternary conjugate according to claim 1, wherein the
imidazoline having NO free radical scavenging activity is
1,3-dioxo-2-[(4-oxyacetoxy)phenyl]-4,4,5,5-tetramethylimidazoline,
the linking arm is L-Lys, L-Asp or L-Glu, the peptide having
thrombolytic activity is an oligopeptide comprising a PAK sequence
(Pro-Ala-Lys), and the thrombus-targeting peptide is an
oligopeptide comprising an RGD sequence (Arg-Gly-Asp).
10. A pharmaceutical composition comprising the ternary conjugate
according to claim 1 and a pharmaceutically acceptable carrier.
11. The pharmaceutical composition according to claim 10, wherein
the ternary conjugate is in the form of a nanospherical
structure.
12. The pharmaceutical composition according to claim 10 for use as
a thrombolytic drug, an NO free radical scavenging drug, or an
anti-thrombus drug.
13. The pharmaceutical composition according to claim 10 for use as
a drug in treating stroke or cerebral infarction.
14. The pharmaceutical composition according to claim 13 for use in
treating stroke or cerebral infarction beyond 3 hours from the
onset of symptoms.
15. A method for preparation of the ternary conjugate according to
claim 1, wherein the ternary conjugate is represented by formula I,
##STR00013## wherein NN represents an imidazoline having NO free
radical scavenging activity; AA.sub.1 represents a linking arm
having at least three groups for linking; AA.sub.2 represents a
peptide having thrombolytic activity; and AA.sub.3 represents a
thrombus-targeting peptide; said method comprising the steps of:
(1) providing the imidazoline having NO free radical scavenging
activity (NN), the linking arm having at least three groups for
linking (AA.sub.1), the peptide having thrombolytic activity
(AA.sub.2) and the thrombus-targeting peptide (AA.sub.3), wherein
the linking arm has a first group for linking, a second group for
linking, and a third group for linking; (2) linking the imidazoline
having NO free radical scavenging activity (NN) to the first group
for linking on the linking arm (AA.sub.1), to form a compound of
general formula IM-1: NN-AA.sub.1 (IM-1); (3) linking the peptide
having thrombolytic activity (AA.sub.2) to the compound of general
formula IM-1, wherein one end of the peptide having thrombolytic
activity is linked to the second group for linking on the linking
arm, to form a compound of general formula IM-2:
NN-AA.sub.1-AA.sub.2 (IM-2); and (4) linking the thrombus-targeting
peptide (AA.sub.3) to the compound of general formula IM-2, wherein
one end of the thrombus-targeting peptide is linked to the third
group for linking on the linking arm, to form the compound of
formula I; wherein the order of steps (3) and (4) may be
reversed.
16. The method according to claim 15, wherein step (1) further
comprises protecting the second and the third groups for linking on
the linking arm (AA.sub.1) with protecting groups, and protecting
active groups of the peptide having thrombolytic activity
(AA.sub.2) and of the thrombus-targeting peptide (AA.sub.3), other
than the end to be used for linking, with protecting groups; step
(3) further comprises deprotecting the protected second group for
linking first, and then linking the peptide having thrombolytic
activity to the deprotected second group for linking; step (4)
further comprises deprotecting the protected third group for
linking first, and then linking the thrombus-targeting peptide to
the deprotected third group for linking; and after step (4), there
is further a step of deprotecting the protected active groups of
the peptide having thrombolytic activity (AA.sub.2) and of the
thrombus-targeting peptide (AA.sub.3).
17. The method according to claim 15, wherein the first group for
linking is an amino group, and the second and the third groups for
linking are selected from the group consisting of a carboxyl group
and an amino group.
18. The method according to claim 17, wherein the linking arm is a
natural amino acid, particularly L-Lys, L-Asp or L-Glu.
19. The method according to claim 17, wherein the peptide having
thrombolytic activity is an oligopeptide comprising a PAK
(Pro-Ala-Lys) sequence, an AKP (Ala-Lys-Pro) sequence or a KAP
(Lys-Ala-Pro) sequence, or a peptide having repeating units of the
PAK sequence, the AKP sequence or the KAP sequence.
20. The method according to claim 17, wherein the
thrombus-targeting peptide is an oligopeptide comprising an RGD
(Arg-Gly-Asp) sequence.
21. The method according to claim 15 or 16, wherein the imidazoline
having NO free radical scavenging activity is
1,3-dioxo-2-[(4-oxyacetoxy)phenyl]-4,4,5,5-tetramethylimidazoline,
the linking arm is L-Lys, L-Asp or L-Glu, the peptide having
thrombolytic activity is an oligopeptide comprising a PAK sequence
(Pro-Ala-Lys), and the thrombus-targeting peptide is an
oligopeptide comprising an RGD sequence (Arg-Gly-Asp).
Description
TECHNICAL FIELD
[0001] The present invention relates to a novel compound
simultaneously having effects of thrombolysis, free radical
scavenging and thrombus-targeting, as well as a preparation method
and use thereof. The present invention further relates to a novel
ternary conjugate of "a peptide comprising a PAK
sequence/imidazoline/a peptide comprising an RGD sequence" formed
by linking together a thrombolytic oligopeptide comprising a PAK
(Pro-Ala-Lys) sequence,
1-(4-oxyacetyl-phenyl)-3,3,4,4-tetramethylimidazo line and a
thrombus-targeting peptide/anti-thrombus oligopeptide comprising an
RGD (Arg-Gly-Asp) sequence via a linking arm containing carboxyl
and amino groups. The present invention further relates to a
pharmaceutical composition comprising the above compound for use in
NO free radical scavenging, thrombolysis, thrombus
targeting/antithrombus therapy, and treatment of stroke/cerebral
infarction. The present invention further relates to a method for
preparation of the compound.
BACKGROUND ART
[0002] Thrombotic diseases rank the first in morbidity and
mortality globally. Coronary artery thrombosis results in
myocardial infarction. Cerebral vascular thrombosis leads to
cerebral infarction, i.e., the clinical ischemic stroke. Patients
with myocardial infarction may be intravenously injected with
thrombolytic agents or have bypass surgeries. It should be noted
that the positive outcome of intravenous injection of thrombolytic
agents to patients with myocardial infarction is
ischemia/reperfusion. Since a large amount of NO free radicals are
generated during the process of ischemia/reperfusion, the
thrombolysis process is associated with myocardial damage and
patient death. This is a serious problem in current thrombolysis
treatment of myocardial infarction. Presently, the treatment of
cerebral infarction is confronted with even more complicated
problems. For example, current thrombolytic agents are all not able
to cross the blood-brain barrier, and therefore the efficacy of
intravenous injection of thrombolytic agents in patients with
cerebral infarction is rather limited. Also, for example, no
appropriate surgical procedure that could save patients with
cerebral infarction is available currently. Similarly, even if
there is a positive outcome from intravenously injecting
thrombolytic agents into patients with cerebral infarction, a
tremendous amount of NO free radicals may still be generated in the
process of ischemia/reperfusion such that the thrombolysis process
is associated with damage of brain tissues and patient death. This
is a serious problem in current thrombolysis treatment of cerebral
infarction. Moreover, four serious problems are present in the
clinical treatment for stroke patients: 1) no medicament other than
tPA (tissue-type plasminogen activator) shows efficacy in stroke
patients; 2) tPA treatment is only effective within 3 hours from
the stroke onset, i.e., there is only a 3-hour window for tPA
treatment; 3) tPA treatment often results in systemic bleeding; 4)
brain tissue damage in patients and patient death associated with
the tremendous amount of NO free radicals produced in the process
of ischemia/reperfusion cannot be avoided by tPA treatment. It is
thus imperative to solve these four problems in order to achieve a
substantive breakthrough in clinical treatment of stroke
patients.
[0003] Two compounds,
N.sup..alpha.-(1,3-dioxo-4,4,5,5-tetramethylimidazoline-2-phenyl-4'-oxyac-
etyl)-n.sup..omega.-fatty acyl-Lys-Arg-Gly-Asp-Val and
N.sup..alpha.-(1,3-dioxo-4,4,5,5-tetramethylimidazoline-2-phenyl-4'-oxyac-
etyl)-n.sup..omega.-fatty acyl-Lys-Arg-Gly-Asp-Phe, are disclosed
in Chinese Patent Publication CN102807604 and CN102807605. Both
compounds are derived from a conjugation of an imidazoline having
NO free radical scavenging activity with an anti-thrombus
oligopeptide comprising an RGD sequence (Arg-Gly-Asp) via lysine.
Unlike the compound of the present invention, these two compounds
do not have a thrombolytic peptide attached therein. These two
compounds do not have a function in thrombolysis, and therefore are
not suitable in the manufacture of thrombolytic medicaments and not
suitable in treatment of patients with ischemic stroke.
[0004] To solve the above problems, there is a need for a new
compound simultaneously having effects of thrombolysis, free
radical scavenging and thrombus-targeting. Furthermore, it is
required that such a new compound is able to be effective even if
administered after 3 hours from the onset of stroke in patients,
i.e., not restricted by the 3-hour window as in the treatment using
tPA; does not cause a systemic bleeding response as in tPA
treatment; and can clear the tremendous amount of NO free radicals
generated during ischemia/reperfusion.
SUMMARY OF THE INVENTION
[0005] The present invention provides a ternary conjugate
simultaneously having activities of crossing blood-brain barrier,
thrombolysis, anti-thrombus and NO free radical scavenging, in
which the three members in the ternary conjugate refer to an
imidazoline having NO free radical scavenging activity, a peptide
having thrombolytic activity, and a thrombus-targeting peptide,
wherein the three members are linked together via a proper linking
arm.
[0006] Specifically, the ternary conjugate of the present invention
may be represented by the compound of formula I:
##STR00001##
wherein, NN represents an imidazoline having NO free radical
scavenging activity; AA.sub.1 represents a linking arm having at
least three groups for linking; AA.sub.2 represents a peptide
having thrombolytic activity; and AA.sub.3 represents a
thrombus-targeting peptide.
[0007] The imidazoline used in the present invention may include
imidazole nitroxyl nitroxide (NN) radicals, which can clear NO and
function to clear oxygen free radicals, providing strong protection
for cells damaged by oxygen free radicals. The imidazoline having
NO free radical scavenging activity according to the present
invention is preferably
1,3-dioxo-2-[(4-oxyacetoxy)phenyl]-4,4,5,5-tetramethylimidazoline,
which has excellent chemical and physical stability, and is not
only suitable for any chemical reaction of conjugating a peptide
having thrombolytic activity with a thrombus-targeting peptide, but
also not susceptible to decomposition during storage, thereby
satisfying the requirements for formulations.
[0008] The linking arm used in the present invention may comprise
at least three groups for linking, e.g., carboxyl and amino groups,
which is used to link the imidazoline, the peptide having
thrombolytic activity, and the thrombus-targeting peptide together.
The linking arm according to the present invention may be natural
amino acids, for example, L-Lys, L-Asp, and L-Glu. When the linking
arm (AA.sub.1) used in the present invention has three or more
groups for linking, one or more NN, AA.sub.2 or AA.sub.3 may be
linked thereby, wherein two or more NN, AA.sub.2 or AA.sub.3 may be
the same or different. For example, when AA.sub.1 has four groups
for linking, one NN, two AA.sub.2 and one AA.sub.3 may be linked
thereby while the two AA.sub.2 may be the same or different
peptides having thrombolytic activity.
[0009] The peptide having thrombolytic activity used in the present
invention may be an oligopeptide comprising a PAK (Pro-Ala-Lys)
sequence, an AKP (Ala-Lys-Pro) sequence or a KAP (Lys-Ala-Pro)
sequence, or a peptide having repeating units of the PAK sequence,
the AKP sequence or the KAP sequence. An oligopeptide refer to a
small-molecule peptide having a molecular weight of 1000 Dalton (D)
or less, which is generally composed of 3 to 8 amino acids. The
oligopeptide having thrombolytic activity according to the present
invention may be a tripeptide to octopeptide that comprises a PAK
sequence, an AKP sequence, or a KAP sequence, preferably a
tripeptide to pentapeptide that comprises a PAK sequence, an AKP
sequence, or a KAP sequence. For instance, the oligopeptide used
for the present invention that comprises a PAK sequence, an AKP
sequence, or a KAP sequence may be PAK, RPAK (Arg-Pro-Ala-Lys),
ARPAK (Ala-Arg-Pro-Ala-Lys), GRPAK (Gly-Arg-Pro-Ala-Lys), QRPAK
(Gln-Arg-Pro-Ala-Lys), AKP, KAP, KPAK (Lys-Pro-Ala-Lys), PAKP
(Pro-Ala-Lys-Pro), AKPAK (Ala-Lys-Pro-Ala-Lys) or PAKPA
(Pro-Ala-Lys-Pro-Ala). For example, the peptide having repeating
units of the PAK sequence, the AKP sequence or the KAP sequence
used in the present invention may be any of those peptides being
described in the Chinese patent publication CN101190941 as a
peptide having thrombolytic activity, including a peptide having
repeating units of the PAK sequence, such as (PAK).sub.2,
(PAK).sub.3, (PAK).sub.4, (PAK).sub.5 and (PAK).sub.6; a peptide
having repeating units of the AKP sequence, such as (AKP).sub.2,
(AKP).sub.3, (AKP).sub.4, (AKP).sub.5 and (AKP).sub.6; and a
peptide having repeating units of the KPA sequence, such as
(KPA).sub.2, (KPA).sub.3, (KPA).sub.4, (KPA).sub.5 and
(KPA).sub.6.
[0010] The thrombus-targeting/anti-thrombus peptide used in the
present invention may be an oligopeptide containing an RGD sequence
(Arg-Gly-Asp). The oligopeptide containing an RGD sequence may be
an RGD-based tetrapeptide, such as RGDS (Arg-Gly-Asp-Ser), RGDV
(Arg-Gly-Asp-Val) and RGDF (Arg-Gly-Asp-Phe). Specific binding of
fibrinogen (Fg) to activated platelet membrane glycoprotein (GP)
IIb/IIIa receptor is the common final pathway leading to platelet
aggregation triggered by various physiological inducers, and plays
an important role in the formation of thrombus. Further, RGD
sequences serve as active sites for the binding of Fg ligands and
activated GPIIb/IIIa receptors and have an activated
platelet-targeting property. Structures comprising an RGD sequence
may competitively inhibit and block the binding of Fg and
GPIIb/IIIa receptors, thereby preventing platelet aggregation and
thrombus formation, so as to enable an RGD-containing oligopeptide
become an effective thrombus-targeting molecule and anti-thrombus
agent.
[0011] Further, the thrombus-targeting peptide used in the present
invention may be any of those polypeptides being described in
Chinese patent publication CN101190940 as a polypeptide having
targeting and anti-thrombus activity, including the polypeptides
obtained from conjugating modification of an RGD peptide with a
YIGS (Tyr-Ile-Gly-Ser) peptide. The polypeptides obtained by
modification includes YIGSRRGDS, YIGSRRGDV, YIGSRRGDF, YIGSRYIGSK,
YIGSRYIGSR, YIGSKRGDS, YIGSKRGDF, YIGSKRGDV, YIGSKYIGSK,
YIGSKYIGSR, RGDSRGDS, RGDVRGDV, RGDFRGDF, RGDSYIGSR, RGDSYIGSK,
RGDVYIGSR, RGDVYIGSK, RGDFYIGSR, or RGDFYIGSK.
[0012] In a preferred embodiment, in the compound according to the
present invention, the imidazoline having NO free radical
scavenging activity is
1,3-dioxo-2-[(4-oxyacetoxy)phenyl]-4,4,5,5-tetramethylimidazo line,
the peptide having thrombolytic activity is an oligopeptide
comprising a PAK sequence (Pro-Ala-Lys), and the thrombus-targeting
peptide is an oligopeptide comprising an RGD sequence
(Arg-Gly-Asp). Thus, the present invention provides a ternary
conjugate of "a peptide comprising a PAK sequence/imidazoline/a
peptide comprising an RGD sequence" simultaneously having
activities in crossing blood-brain barrier, thrombolysis,
anti-thrombus and NO free radical scavenging.
[0013] In an embodiment, in the compound according to the present
invention, the imidazoline having NO free radical scavenging
activity is
1,3-dioxo-2-[(4-oxyacetoxy)phenyl]-4,4,5,5-tetramethylimidazoline,
the linking arm is L-Lys, the peptide having thrombolytic activity
is an oligopeptide comprising a PAK sequence (Pro-Ala-Lys), and the
thrombus-targeting peptide is an oligopeptide comprising an RGD
sequence (Arg-Gly-Asp). In this case, the oligopeptide comprising a
PAK sequence may be an ARPAK pentapeptide, a GRPAK pentapeptide, an
RPAK tetrapeptide, or a PAK tripeptide; the oligopeptide comprising
an RGD sequence (Arg-Gly-Asp) may be an RGD-based tetrapeptide,
such as RGDS, RGDV or RGDF. When L-Lys is used as the linking arm,
the compound according to the present invention may be of following
general formula I-1 or I-2:
##STR00002##
wherein, aa.sub.1 and aa.sub.2 may be both present or both absent,
or aa.sub.1 is present but aa.sub.2 is absent; when both of
aa.sub.1 and aa.sub.2 are present, aa.sub.1 is R (Arg), and
aa.sub.2 is G (Gly), A (Ala) or Q (Gln); when aa.sub.1 is present
but aa.sub.2 is absent, aa.sub.1 is R (Arg); aa.sub.3 may be S
(Ser), V (Val), or F (Phe).
[0014] For examples related to the compound of general formula I-1,
in a preferred example, the compound according to the present
invention may be a ternary conjugate of ARPAK/imidazoline/RGD
represented by following formula I-1-1; in another preferred
example, the compound according to the present invention may be a
ternary conjugate of GRPAK/imidazoline/RGD represented by following
formula I-1-2; in still another preferred example, the compound
according to the present invention may be a ternary conjugate of
RPAK/imidazoline/RGD represented by following formula I-1-3; and in
still another preferred example, the compound according to the
present invention may be a ternary conjugate of PAK/imidazoline/RGD
represented by following formula I-1-4:
##STR00003##
wherein aa.sub.3 may be S (Ser), V (Val) or F (Phe), preferably V
(Val).
[0015] For examples related to the compound of general formula I-2,
the compound according to the present invention may be preferably
of following general formula I-2-1, I-2-2, I-2-3 or I-2-4:
##STR00004##
wherein aa.sub.3 may be S (Ser), V (Val) or F (Phe), preferably V
(Val).
[0016] In another embodiment, in the compound according to the
present invention, the imidazoline having NO free radical
scavenging activity is
1,3-dioxo-2-[(4-oxyacetoxy)phenyl]-4,4,5,5-tetramethylimidazoline,
the linking arm is L-Asp, the peptide having thrombolytic activity
is an oligopeptide comprising a PAK sequence (Pro-Ala-Lys), and the
thrombus-targeting peptide is an oligopeptide comprising an RGD
sequence (Arg-Gly-Asp). When L-Asp is used as the linking arm, the
compound according to the present invention may be of following
general formula I-3 or I-4:
##STR00005##
wherein, aa.sub.1 and aa.sub.2 may be both present or both absent,
or aa.sub.1 is present but aa.sub.2 is absent; when both of
aa.sub.1 and aa.sub.2 are present, aa.sub.1 is R (Arg), and
aa.sub.2 is G (Gly), A (Ala) or Q (Gln); when aa.sub.1 is present
but aa.sub.2 is absent, aa.sub.1 is R (Arg); aa.sub.3 may be S
(Ser), V (Val), or F (Phe). aa.sub.1 is preferably R (Arg),
aa.sub.2 is preferably G (Gly), and aa.sub.3 is preferably V
(Val).
[0017] For examples related to the compound of general formula I-3,
the compound according to the present invention may be preferably
of following general formula I-3-1, I-3-2, I-3-3 or I-3-4:
##STR00006##
wherein aa.sub.3 may be S (Ser), V (Val) or F (Phe), preferably V
(Val).
[0018] For examples related to the compound of general formula I-4,
the compound according to the present invention may be preferably
of following general formula I-4-1, I-4-2, I-4-3 or I-4-4:
##STR00007##
wherein aa.sub.3 may be S (Ser), V (Val) or F (Phe), preferably V
(Val).
[0019] In still another embodiment, in the compound according to
the present invention, the imidazoline having NO free radical
scavenging activity is
1,3-dioxo-2-[(4-oxyacetoxy)phenyl]-4,4,5,5-tetramethylimidazoline,
the linking arm is L-Glu, the peptide having thrombolytic activity
is an oligopeptide comprising a PAK sequence (Pro-Ala-Lys), and the
thrombus-targeting peptide is an oligopeptide comprising an RGD
sequence (Arg-Gly-Asp). When L-Glu is used as the linking arm, the
compound according to the present invention may be of following
general formula I-5 or I-6:
##STR00008##
wherein, aa.sub.1 and aa.sub.2 may be both present or both absent,
or aa.sub.1 is present but aa.sub.2 is absent; when both of
aa.sub.1 and aa.sub.2 are present, aa.sub.1 is R (Arg), and
aa.sub.2 is G (Gly), A (Ala) or Q (Gln); when aa.sub.1 is present
but aa.sub.2 is absent, aa.sub.1 is R (Arg); aa.sub.3 may be S
(Ser), V (Val), or F (Phe). aa.sub.1 is preferably R (Arg),
aa.sub.2 is preferably G (Gly), and aa.sub.3 is preferably V
(Val).
[0020] For examples related to the compound of general formula I-5,
the compound according to the present invention may be preferably
of following general formula I-5-1, I-5-2, I-5-3 or I-5-4:
##STR00009##
wherein aa.sub.3 may be S (Ser), V (Val) or F (Phe), preferably V
(Val).
[0021] For examples related to the compound of general formula I-6,
the compound according to the present invention may be preferably
of following general formula I-6-1, I-6-2, I-6-3 or I-6-4:
##STR00010##
wherein aa.sub.3 may be S (Ser), V (Val) or F (Phe), preferably V
(Val).
[0022] In another aspect, the present invention further relates to
a pharmaceutical composition comprising the above compound
according to the present invention and a pharmaceutically
acceptable carrier. Preferably, the pharmaceutical composition
according to the present invention comprises the compound of above
general formula I-1, I-2, I-3, I-4, I-5 or I-6. More preferably,
the pharmaceutical composition according to the present invention
comprises the compound of above general formula I-1-1, I-1-2, I-1-3
or I-1-4. When the pharmaceutical composition according to the
present invention comprises the compound of the general formula
I-1-1, I-1-2, I-1-3 or I-1-4, the compound may be in the form of a
dimer, trimer or tetramer structure in the pharmaceutical
composition, and may be in the form of a nanosphere having a
diameter of 2 to 300 nm. In the pharmaceutical composition
according to the present invention, the nanospherical structure may
preferably have a diameter of 2 to 100 nm. It is a fact well known
in nanopharmacology that nanospheres having a diameter of less than
100 nm are less prone to be engulfed by macrophages during
transportation in blood and may readily cross the blood capillary
wall. These properties allow the compound according to the present
invention to cross the blood-brain barrier. The pharmaceutical
composition according to the present invention may be used as a
thrombolytic drug in treating diseases such as myocardial
infarction, ischemic stroke, deep vein thrombosis, pulmonary
embolism, peripheral arterial occlusive disease, occluded central
vascular access devices, clotted arteriovenous fistula and shunts,
and carotid stenosis. The pharmaceutical composition according to
the present invention may also be used as an NO free
radical-scavenging drug in treating neurodegenerative diseases,
such as Alzheimer's disease, Parkinson's disease, motor neuron
diseases, amyotrophic lateral sclerosis, noise-induced hearing
loss, Lou Gehrig's disease or Huntington's disease; in treating
cardiovascular diseases, such as atherosclerosis, coronary heart
disease or myocardial infarction; in treating mental diseases, such
as bipolar disorder, schizophrenia or autism; and in treating
diseases including altitude sickness, diabetes, rheumatoid
arthritis, traumatic brain injury, cancer, fragile X syndrome,
sickle cell disease, Lichen planus, vitiligo, chronic fatigue
syndrome and so on. The pharmaceutical composition according to the
present invention may further be used as a thrombus
targeting/anti-thrombus drug in treating diseases such as
thrombocytosis, myeloproliferative disease, polycythemia vera or
Budd-Chiari syndrome. The pharmaceutical composition according to
the present invention may also be used as a drug in treating stroke
or cerebral infarction, preferably in treating stroke or cerebral
infarction beyond 3, 4, 6 and 24 hours from the onset of symptoms
with successive administrations. The pharmaceutical
composition/compound according to the present invention
simultaneously has functions of NO free radical scavenging,
thrombolysis, and anti-thrombus/thrombus targeting, and therefore
shows efficacy even when being administered after 3 hours from the
onset of stroke in patients; namely, it is not restricted by the
3-hour window as in the treatment using tPA, does not cause a
systemic bleeding response as tPA, and can clear the tremendous
amount of NO free radicals generated during ischemia/reperfusion,
preventing damage to cranial nerve tissues in patients during the
treatment. In the pharmaceutical composition according to the
present invention, the nanospherical structures of the compounds
are able to maximize the effects of blood-brain barrier crossing,
thrombolysis, thrombus targeting/anti-thrombus, as well as the
effect of clearing the NO free radicals generated during
ischemia/reperfusion.
[0023] The pharmaceutical composition according to the present
invention may be any clinically acceptable formulation, for
example, an injectable formulation (powder for injection,
lyophilized powder for injection, liquid for injection, infusion
etc.), a tablet, oral liquid, a granule, a capsule, a soft capsule,
a dripping pill and so on, wherein the pharmaceutically acceptable
carriers may be one or more of xylitol, manitol, lactose, fructose,
dextran, glucose, polyvinylpyrrolidone, low-molecular-weight
dextran, sodium chloride, calcium gluconate, or calcium phosphate.
In addition, the pharmaceutical composition according to the
present invention may further comprise an excipient that may be an
antioxidant complexing agent, a filler, a framework material, and
so on.
[0024] In another aspect, the present invention further relates to
a preparation method of the aforementioned compound of formula I,
comprising the steps of:
(1) providing an imidazoline having NO free radical scavenging
activity (NN), a linking arm having at least three groups for
linking (AA.sub.1), a peptide having thrombolytic activity
(AA.sub.2) and a thrombus-targeting peptide (AA.sub.3), wherein the
linking arm has a first group for linking, a second group for
linking, and a third group for linking; (2) under appropriate
reaction conditions, linking the imidazoline having NO free radical
scavenging activity (NN) to the first group for linking on the
linking arm (AA.sub.1), to form a compound of general formula
IM-1:
NN-AA.sub.1 (IM-1);
(3) under appropriate reaction conditions, linking the peptide
having thrombolytic activity (AA.sub.2) to the compound of general
formula IM-1, wherein one end of the peptide having thrombolytic
activity is linked to the second group for linking on the linking
arm, to form a compound of general formula IM-2:
NN-AA.sub.1-AA.sub.2 (IM-2); and
(4) under appropriate reaction conditions, linking the
thrombus-targeting peptide (AA.sub.3) to the compound of general
formula IM-2, wherein one end of the thrombus-targeting peptide is
linked to the third group for linking on the linking arm, to form
the compound of formula I; wherein step (3) and (4) are
exchangeable in order.
[0025] In the preparation method according to the present
invention, step (1) further comprises protecting the second and the
third groups for linking on the linking arm (AA.sub.1) with
protecting groups, and protecting active groups of the peptide
having thrombolytic activity (AA.sub.2) and of the
thrombus-targeting peptide (AA.sub.3), other than the end to be
used for linking, with protecting groups; step (3) further
comprises deprotecting the protected second group for linking
first, and then linking the peptide having thrombolytic activity to
the deprotected second group for linking; step (4) further
comprises deprotecting the protected third group for linking first,
and then linking the thrombus-targeting peptide to the deprotected
third group for linking; and after step (4), there is further a
step of deprotecting the protected active groups of the peptide
having thrombolytic activity (AA.sub.2) and of the
thrombus-targeting peptide (AA.sub.3). By applying techniques of
adding and removing protecting groups, the order in which NN,
AA.sub.2 and AA.sub.3 are linked to the linking arm and linking
position thereof are controllable. Protecting groups on other
active groups are then removed after completion of the coupling.
Appropriate reaction conditions refer to conventional conditions
employed in peptide synthesis. The imidazoline having NO free
radical scavenging activity (NN), the linking arm having at least
three groups for linking (AA.sub.1), the peptide having
thrombolytic activity (AA.sub.2), and the thrombus-targeting
peptide (AA.sub.3) are the same as defined above for the compound
of formula I according to the present invention.
[0026] The preparation method of the present invention may be
further understood from the more detailed description as
follows.
[0027] In an embodiment, the first group for linking on the linking
arm in the preparation method according to the present invention is
an amino group, while the second and the third groups for linking
are selected from the group consisting of a carboxyl group and an
amino group.
[0028] In a preferred embodiment of the preparation method
according to the present invention, the imidazoline having NO free
radical scavenging activity is
1,3-dioxo-2-[(4-oxyacetoxy)phenyl]-4,4,5,5-tetramethylimidazoline,
the linking arm is L-Lys, the peptide having thrombolytic activity
is an oligopeptide comprising a PAK sequence (Pro-Ala-Lys), and the
thrombus-targeting peptide is an oligopeptide comprising an RGD
sequence (Arg-Gly-Asp). When the linking arm is L-Lys, there may be
the following two ways for conjugating: [0029] (1)
1,3-dioxo-2-[(4-oxyacetoxy)phenyl]-4,4,5,5-tetramethylimidazoline
is linked to an amino group on the L-Lys linking arm, a carboxyl
group on the oligopeptide comprising a PAK sequence is linked to
another amino group on the L-Lys linking arm, and an amino group on
the oligopeptide comprising an RGD sequence is linked to a carboxyl
group on the L-Lys linking arm (as shown in the above compound of
formula I-1); or [0030] (2)
1,3-dioxo-2-[(4-oxyacetoxy)phenyl]-4,4,5,5-tetramethylimidazoline
is linked to an amino group on the L-Lys linking arm, an amino
group on the oligopeptide comprising a PAK sequence is linked to a
carboxyl group on the L-Lys linking arm, and a carboxyl group on
the oligopeptide comprising an RGD sequence is linked to another
amino group on the L-Lys linking arm (as shown in the above
compound of formula I-2).
[0031] For examples related to the compound of formula I-1, when
the compound of general formula I-1-1, I-1-2, I-1-3 or I-1-4 is
prepared, the preparation method of the present invention may be
carried out according to the synthesis schemes shown in FIGS. 1 to
4. FIG. 1 shows a synthesis scheme for the compound of general
formula I-1-1. FIG. 2 shows a synthesis scheme for the compound of
general formula I-1-2. FIG. 3 shows a synthesis scheme for the
compound of general formula I-1-3. FIG. 4 shows a synthesis scheme
for the compound of general formula I-1-4. In FIGS. 1 to 4,
aa.sub.3 may be S (Ser), V (Val), or F (Phe), as described above.
For examples related to the compound of general formula I-1-2, the
preparation method according to the present invention is described
as follows:
(1) preparing
1,3-dioxo-2-(4-oxyacetoxy-phenyl)-4,4,5,5-tetramethylimidazoline;
(2) preparing
1,3-dioxo-2-[(4'-oxyacetyl-Lys-OMe)phenyl]-4,4,5,5-tetramethyli-
midazoline (the carboxyl group on the Lys linking arm is protected
with a protecting group); (3) preparing
HCl.Arg(NO.sub.2)-Gly-Asp(OBzl)-Ser(Bzl)-Obzl,
HCl.Arg(NO.sub.2)-Gly-Asp(OBzl)-Val-Obzl or
HCl.Arg(NO.sub.2)-Gly-Asp(OBzl)-Phe-Obzl; [0032] (4) preparing
Boc-Gly-Arg(NO.sub.2)-Pro-Ala-Lys(Z); [0033] (5) linking
Boc-Gly-Arg(NO.sub.2)-Pro-Ala-Lys(Z) to the lysine of
1,3-dioxo-2-[(4'-oxyacetyl-Lys-OMe)phenyl]-4,4,5,5-tetramethylimidazoline
to provide
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Gly-Arg(NO.sub.2)-Pro-Ala-L-
ys(Z)]-Lys}phenyl}-4,4,5,5-tetramethylimidazoline; [0034] (6)
respectively conjugating
HCl.Arg(NO.sub.2)-Gly-Asp(OBzl)-Ser(Bzl)-Obzl,
HCl.Arg(NO.sub.2)-Gly-Asp (OBzl)-Val-Obzl, or HCl.Arg
(NO.sub.2)-Gly-Asp(OBzl)-Phe-Obzl to
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Gly-Arg(NO.sub.2)-Pro-Ala-L-
ys (Z)]-Lys}phenyl}-4,4,5,5-tetramethylimidazoline to afford
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Gly-Arg(NO.sub.2)-Pro-Ala-L-
ys (Z)]-Lys-Arg(NO.sub.2)-Gly-Asp
(OBzl)-Ser(Bzl)-OBzl}phenyl}-4,4,5,5-tetramethylimidazoline,
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Gly-Arg(NO.sub.2)-Pro-Ala-L-
ys(Z)]-Lys-Arg-(NO.sub.2)-Gly-Asp-(OBzl)-Val-OBzl}phenyl}-4,4,5,5-tetramet-
hylimidazoline, or
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Gly-Arg(NO.sub.2)-Pro-Ala-L-
ys
(Z)]-Lys-Arg(NO.sub.2)-Gly-Asp(OBzl)-Phe-OBzl}phenyl}-4,4,5,5-tetrameth-
ylimidazoline, respectively; [0035] (7) deprotecting the compounds
resulting from step (6) to afford
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Gly-Arg-Pro-Ala-Lys]-Lys-Arg-Gl-
y-Asp-Ser}phenyl}-4,4,5,5-tetramethylimidazolin,
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Gly-Arg-Pro-Ala-Lys]-Lys-Arg-Gl-
y-Asp-Val}phenyl}-4,4,5,5-tetramethylimidazoline or
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Gly-Arg-Pro-Ala-Lys]-Lys-Arg-Gl-
y-Asp-Phe}phenyl}-4,4,5,5-tetramethylimidazoline.
[0036] In the case that the compounds of general formula I-1-1,
I-1-3 and I-1-4 are prepared, the above manufacturing process is
repeated but replacing "Boc-Gly-Arg(NO.sub.2)-Pro-Ala-Lys(Z)" in
step (4) with "Boc-Ala-Arg(NO.sub.2)-Pro-Ala-Lys(Z)",
"Boc-Arg(NO.sub.2)-Pro-Ala-Lys(Z)" and "Boc-Pro-Ala-Lys(Z)".
[0037] Active groups at appropriate positions on the oligopeptide
comprising a PAK sequence and the oligopeptide comprising an RGD
sequence may be protected per need of conjugation design, so that
one end of the selected sequences (comprising an active group to be
attached to the linking arm) is used to couple to an active group
on the linking arm. The step of coupling the oligopeptide
comprising a PAK sequence and the step of coupling the oligopeptide
comprising an RGD sequence are exchangeable in order. For example,
the oligopeptide comprising an RGD sequence is coupled to the
linking arm first, and then the oligopeptide comprising a PAK
sequence is coupled thereto.
[0038] Active groups include groups that may be subjected to
condensation reaction, such as an amino group or a carboxyl group.
Amino-protecting groups may be carboxybenzyl (CBz), t-butoxy
carbonyl (Boc), 9-florenyl methoxy carbonyl (Fmoc), benzyl (Bn) or
p-methoxyphenyl (PMP). Carboxyl-protecting groups may be methyl
ester (OMe), benzyl ester (OBn), benzyl methyl ester (Obzl),
t-butyl ester (OBUT), or silyl ester (OSi(CH.sub.3).sub.3).
[0039] For examples related to the compound of formula I-2, when
the compound of general formula I-2-1, I-2-2, I-2-3 or I-2-4 is
prepared, the preparation method of the present invention may be
carried out according to the synthesis schemes shown in FIGS. 5 to
8. FIG. 5 shows a synthesis scheme for the compound of general
formula I-2-1. FIG. 6 shows a synthesis scheme for the compound of
general formula I-2-2. FIG. 7 shows a synthesis scheme for the
compound of general formula I-2-3. FIG. 8 shows a synthesis scheme
for the compound of general formula I-2-4. In FIGS. 5 to 8,
aa.sub.3 may be S (Ser), V (Val), or F (Phe), as described above.
When the compound of general formula I-2-1, I-2-2, I-2-3 or I-2-4
is prepared,
1,3-dioxo-2-[(4'-oxyacetyl-Lys-OMe)phenyl]-4,4,5,5-tetramethylimidazoline
may be prepared first, and then the C terminal of the oligopeptide
comprising an RGD sequence is attached to the amino group on the
Lys linking arm; and finally, the N terminal of the oligopeptide
comprising a PAK sequence is attached to the deprotected carboxyl
group on the linking arm.
[0040] In another embodiment, in the preparation method according
to the present invention, the imidazoline having NO free radical
scavenging activity is
1,3-dioxo-2-[(4-oxyacetoxy)phenyl]-4,4,5,5-tetramethylimidazoline,
the linking arm is L-Asp, the peptide having thrombolytic activity
is an oligopeptide comprising a PAK sequence (Pro-Ala-Lys), and the
thrombus-targeting peptide is an oligopeptide comprising an RGD
sequence (Arg-Gly-Asp), wherein
1,3-dioxo-2-[(4-oxyacetoxy)phenyl]-4,4,5,5-tetramethylimidazoline
is linked to the amino group on the L-Asp linking arm, the amino
group on the oligopeptide comprising a PAK sequence is linked to
one carboxyl group on the L-Asp linking arm, and the amino group on
the oligopeptide comprising an RGD sequence is linked to another
carboxyl group on the L-Asp linking arm (as shown in the above
compound of formula I-3 or I-4).
[0041] For examples related to the compound of formula I-3, when
the compound of general formula I-3-1, I-3-2, I-3-3 or I-3-4 is
prepared, the preparation method of the present invention may be
carried out according to the synthesis schemes shown in FIGS. 9 to
12. FIG. 9 shows a synthesis scheme for the compound of general
formula I-3-1. FIG. 10 shows a synthesis scheme for the compound of
general formula I-3-2. FIG. 11 shows a synthesis scheme for the
compound of general formula I-3-3. FIG. 12 shows a synthesis scheme
for the compound of general formula I-3-4. In FIGS. 9 to 12,
aa.sub.3 may be S (Ser), V (Val), or F (Phe), as described above.
When the compound of general formula I-3-1, I-3-2, I-3-3 or I-3-4
is prepared,
1,3-dioxo-2-[(4'-oxyacetyl-Asp-OMe)phenyl]-4,4,5,5-tetramethylimidazoline
may be prepared first, and then the N terminal of the oligopeptide
comprising a PAK sequence is attached to one carboxyl group on the
Asp linking arm; and finally, the N terminal of the oligopeptide
comprising an RGD sequence is attached to another deprotected
carboxyl group on the Asp linking arm.
[0042] For examples related to the compound of formula I-4, when
the compound of general formula I-4-1, I-4-2, I-4-3 or I-4-4 is
prepared, the preparation method of the present invention may be
carried out according to the synthesis schemes shown in FIGS. 13 to
16. FIG. 13 shows a synthesis scheme for the compound of general
formula I-4-1. FIG. 14 shows a synthesis scheme for the compound of
general formula I-4-2. FIG. 15 shows a synthesis scheme for the
compound of general formula I-4-3. FIG. 16 shows a synthesis scheme
for the compound of general formula I-4-4. In FIGS. 13 to 16,
aa.sub.3 may be S (Ser), V (Val), or F (Phe), as described above.
When the compound of general formula I-4-1, I-4-2, I-4-3 or I-4-4
is prepared,
1,3-dioxo-2-[(4'-oxyacetyl-Asp-OMe)phenyl]-4,4,5,5-tetramethylimidazoline
may be prepared first, and then the N terminal of the oligopeptide
comprising an RGD sequence is attached to one carboxyl group on the
Asp linking arm; and finally, the N terminal of the oligopeptide
comprising a PAK sequence is attached to another deprotected
carboxyl group on the Asp linking arm.
[0043] In still another embodiment, in the preparation method
according to the present invention, the imidazoline having NO free
radical scavenging activity is
1,3-dioxo-2-[(4-oxyacetoxy)phenyl]-4,4,5,5-tetramethylimidazoline,
the linking arm is L-Glu, the peptide having thrombolytic activity
is an oligopeptide comprising a PAK sequence (Pro-Ala-Lys), and the
thrombus-targeting peptide is an oligopeptide comprising an RGD
sequence (Arg-Gly-Asp), wherein
1,3-dioxo-2-[(4-oxyacetoxy)phenyl]-4,4,5,5-tetramethylimidazoline
is linked to the amino group on the L-Glu linking arm, the amino
group on the oligopeptide comprising a PAK sequence is linked to
one carboxyl group on the L-Glu linking arm, and the amino group on
the oligopeptide comprising an RGD sequence is linked to another
carboxyl group on the L-Glu linking arm (as shown in the above
compound of formula I-5 or I-6).
[0044] For examples related to the compound of formula I-5, when
the compound of general formula I-5-1, I-5-2, I-5-3 or I-5-4 is
prepared, the preparation method of the present invention may be
carried out according to the synthesis schemes shown in FIGS. 17 to
20. FIG. 17 shows a synthesis scheme for the compound of general
formula I-5-1. FIG. 18 shows a synthesis scheme for the compound of
general formula I-5-2. FIG. 19 shows a synthesis scheme for the
compound of general formula I-5-3. FIG. 20 shows a synthesis scheme
for the compound of general formula I-5-4. In FIGS. 17 to 20,
aa.sub.3 may be S (Ser), V (Val), or F (Phe), as described above.
When the compound of general formula I-5-1, I-5-2, I-5-3 or I-5-4
is prepared,
1,3-dioxo-2-[(4'-oxyacetyl-Glu-OMe)phenyl]-4,4,5,5-tetramethylimidazoline
may be prepared first, and then the N terminal of the oligopeptide
comprising an RGD sequence is attached to one carboxyl group on the
Glu linking arm; and finally, the N terminal of the oligopeptide
comprising an PAK sequence is attached to another deprotected
carboxyl group on the Glu linking arm.
[0045] For examples related to the compound of formula I-6, when
the compound of general formula I-6-1, I-6-2, I-6-3 or I-6-4 is
prepared, the preparation method of the present invention may be
carried out according to the synthesis schemes shown in FIGS. 21 to
24. FIG. 21 shows a synthesis scheme for the compound of general
formula I-6-1. FIG. 22 shows a synthesis scheme for the compound of
general formula I-6-2. FIG. 23 shows a synthesis scheme for the
compound of general formula I-6-3. FIG. 24 shows a synthesis scheme
for the compound of general formula I-6-4. In FIGS. 21 to 24,
aa.sub.3 may be S (Ser), V (Val), or F (Phe), as described above.
When the compound of general formula I-6-1, I-6-2, I-6-3 or I-6-4
is prepared,
1,3-dioxo-2-[(4'-oxyacetyl-Glu-OMe)phenyl]-4,4,5,5-tetramethylimidazoline
may be prepared first, and then the N terminal of the oligopeptide
comprising a PAK sequence is attached to one carboxyl group on the
Glu linking arm; and finally, the N terminal of the oligopeptide
comprising an RGD sequence is attached to another deprotected
carboxyl group on the Glu linking arm.
[0046] In the preparation method previously described, the
oligopeptide comprising a PAK sequence may be
ARPAK(Ala-Arg-Pro-Ala-Lys), GRPAK(Gly-Arg-Pro-Ala-Lys),
QRPAK(Gln-Arg-Pro-Ala-Lys), RPAK(Arg-Pro-Ala-Lys) or
PAK(Pro-Ala-Lys), and the oligopeptide comprising an RGD sequence
may be RGDS(Arg-Gly-Asp-Ser), RGDV(Arg-Gly-Asp-Val) or
RGDF(Arg-Gly-Asp-Phe).
[0047] For the compound or pharmaceutical composition according to
the present invention, high NO free radical-scavenging activity is
demonstrated by in vivo rat models of NO free radical scavenging;
superior thrombolysis and anti-thrombus activities are demonstrated
by in vivo and in vitro experiments of thrombolysis and
anti-thrombus; neuroprotective efficacy and superior anti-stroke
activity are demonstrated by in vivo rat stroke models; and
efficacy in decreasing cerebral infarction volume is demonstrated
by rat stroke models.
DESCRIPTION OF DRAWINGS
[0048] FIG. 1 shows a synthesis scheme for an embodiment of the
compound according to the present invention (the compound of
general formula I-1-1);
[0049] FIG. 2 shows a synthesis scheme for an embodiment of the
compound according to the present invention (the compound of
general formula I-1-2);
[0050] FIG. 3 shows a synthesis scheme for an embodiment of the
compound according to the present invention (the compound of
general formula I-1-3);
[0051] FIG. 4 shows a synthesis scheme for an embodiment of the
compound according to the present invention (the compound of
general formula I-1-4);
[0052] FIG. 5 shows a synthesis scheme for an embodiment of the
compound according to the present invention (the compound of
general formula I-2-1);
[0053] FIG. 6 shows a synthesis scheme for an embodiment of the
compound according to the present invention (the compound of
general formula I-2-2);
[0054] FIG. 7 shows a synthesis scheme for an embodiment of the
compound according to the present invention (the compound of
general formula I-2-3);
[0055] FIG. 8 shows a synthesis scheme for an embodiment of the
compound according to the present invention (the compound of
general formula I-2-4);
[0056] FIG. 9 shows a synthesis scheme for an embodiment of the
compound according to the present invention (the compound of
general formula I-3-1);
[0057] FIG. 10 shows a synthesis scheme for an embodiment of the
compound according to the present invention (the compound of
general formula I-3-2);
[0058] FIG. 11 shows a synthesis scheme for an embodiment of the
compound according to the present invention (the compound of
general formula I-3-3);
[0059] FIG. 12 shows a synthesis scheme for an embodiment of the
compound according to the present invention (the compound of
general formula I-3-4);
[0060] FIG. 13 shows a synthesis scheme for an embodiment of the
compound according to the present invention (the compound of
general formula I-4-1);
[0061] FIG. 14 shows a synthesis scheme for an embodiment of the
compound according to the present invention (the compound of
general formula I-4-2);
[0062] FIG. 15 shows a synthesis scheme for an embodiment of the
compound according to the present invention (the compound of
general formula I-4-3);
[0063] FIG. 16 shows a synthesis scheme for an embodiment of the
compound according to the present invention (the compound of
general formula I-4-4);
[0064] FIG. 17 shows a synthesis scheme for an embodiment of the
compound according to the present invention (the compound of
general formula I-5-1);
[0065] FIG. 18 shows a synthesis scheme for an embodiment of the
compound according to the present invention (the compound of
general formula I-5-2);
[0066] FIG. 19 shows a synthesis scheme for an embodiment of the
compound according to the present invention (the compound of
general formula I-5-3);
[0067] FIG. 20 shows a synthesis scheme for an embodiment of the
compound according to the present invention (the compound of
general formula I-5-4);
[0068] FIG. 21 shows a synthesis scheme for an embodiment of the
compound according to the present invention (the compound of
general formula I-6-1);
[0069] FIG. 22 shows a synthesis scheme for an embodiment of the
compound according to the present invention (the compound of
general formula I-6-2);
[0070] FIG. 23 shows a synthesis scheme for an embodiment of the
compound according to the present invention (the compound of
general formula I-6-3);
[0071] FIG. 24 shows a synthesis scheme for an embodiment of the
compound according to the present invention (the compound of
general formula I-6-4);
[0072] FIG. 25 shows the nanostructures of compound Ia according to
the present invention in 1.times.10.sup.-6 M, 1.times.10.sup.-9 M
and 1.times.10.sup.-12 M aqueous solutions;
[0073] FIG. 26 shows the nanostructures of compound Ib according to
the present invention in 1.times.10.sup.-6 M, 1.times.10.sup.-9 M
and 1.times.10.sup.-12 M aqueous solutions;
[0074] FIG. 27 shows the nanostructures of compound Ic according to
the present invention in 1.times.10.sup.-6 M, 1.times.10.sup.-9 M
and 1.times.10.sup.-12 M aqueous solutions;
[0075] FIG. 28 shows the nanostructures of compound Id according to
the present invention in 1.times.10.sup.-6 M, 1.times.10.sup.-9 M
and 1.times.10.sup.-12 M aqueous solutions;
[0076] FIG. 29 shows the nanostructures of compound Ie according to
the present invention in 1.times.10.sup.-6 M, 1.times.10.sup.-9 M
and 1.times.10.sup.-12 M aqueous solutions;
[0077] FIG. 30 shows the nanostructures of compound If according to
the present invention in 1.times.10.sup.-6M, 1.times.10.sup.-9 M
and 1.times.10.sup.-12 M aqueous solutions;
[0078] FIG. 31 shows the nanostructures of compound Ig according to
the present invention in 1.times.10.sup.-6M, 1.times.10.sup.-9 M
and 1.times.10.sup.-12 M aqueous solutions;
[0079] FIG. 32 shows the nanostructures of compound Ih according to
the present invention in 1.times.10.sup.-6M, 1.times.10.sup.-9 M
and 1.times.10.sup.-12 M aqueous solutions;
[0080] FIG. 33 shows the nanostructures of compound Ii according to
the present invention in 1.times.10.sup.-6M, 1.times.10.sup.-9 M
and 1.times.10.sup.-12 M aqueous solutions;
[0081] FIG. 34 shows the nanostructures of compound Ij according to
the present invention in 1.times.10.sup.-6M, 1.times.10.sup.-9 M
and 1.times.10.sup.-12 M aqueous solutions;
[0082] FIG. 35 shows the nanostructures of compound Ik according to
the present invention in 1.times.10.sup.-6M, 1.times.10.sup.-9 M
and 1.times.10.sup.-12 M aqueous solutions;
[0083] FIG. 36 shows the nanostructures of compound Il according to
the present invention in 1.times.10.sup.-6M, 1.times.10.sup.-9 M
and 1.times.10.sup.-12 M aqueous solutions;
[0084] FIG. 37 shows the high-resolution FT-MS spectrum of compound
Ie according to the present invention at a concentration of 0.01
.mu.M;
[0085] FIG. 38 shows the high-resolution FT-MS spectrum of compound
Ie according to the present invention at a concentration of 0.1
.mu.M;
[0086] FIG. 39 shows the high-resolution FT-MS spectrum of compound
Ie according to the present invention at a concentration of 1
.mu.M;
[0087] FIG. 40 shows the high-resolution FT-MS spectrum of compound
Ie according to the present invention at a concentration of 10
.mu.M.
DETAILED DESCRIPTION OF EMBODIMENTS
[0088] The present invention will now be described in connection
with the following specific examples, and the advantages and
features thereof will become apparent in view of the description.
These examples are merely illustrative and in no way limit the
scope of the present invention. A person skilled in the art can
understand that modification or substitution may be made in details
and formality of the technical solutions of the present invention
without deviating from the spirit and scope of the present
invention, and these modifications or substitutions are intended to
be within the scope of protection of the present invention.
Preparation of Imidazolines Having NO Free Radical Scavenging
Activity:
1,3-dioxo-2-[(4-oxyacetoxy)phenyl]-4,4,5,5-tetramethylimidazoline
Example 1
Preparation of 2,3-dimethyl-2,3-dinitrobutane
[0089] 69 g (0.78 mol) 2-nitropropane was added to a 130 ml aqueous
solution of NaOH (6N). 20 ml (0.38 mol) Br.sub.2 was added dropwise
under stirring on an ice salt bath within 1 h. After completion of
Br.sub.2 addition, 240 mL ethanol was added thereto and refluxed at
90.degree. C. for 3 h. The reaction solution, while still hot, was
instantly poured into 800 ml ice water, and then filtered to afford
55 g of the title compound (81%) as a colorless flaky crystal, Mp
110-112.degree. C.
Example 2
Preparation of 2,3-dimethyl-2,3-dihydroxyaminobutane
[0090] 7 g (40 mmol) 2,3-dimethyl-2,3-dinitrobutane and 4 g
NH.sub.4Cl were mixed and suspended in a 80 mL aqueous solution of
ethanol (50%) and stirred on ice bath, into which 16 g zinc powder
was added within 3 h. After the addition of zinc powder was
completed, the ice bath was removed, and the reaction continued for
3 h at room temperature (RT) under stirring, and then the reaction
mixture was vacuum filtered. The filter cake was washed repeatedly
with ethanol aqueous solution (50%). The filtrate and the washing
liquid were combined, adjusted to pH=2 with conc. HCl, and then
distilled under reduced pressure into a slurry. After addition of
an appropriate amount of potassium carbonate, the slurry was evenly
mixed and extracted for 6 h by using a Soxhlet extractor with
chloroform as the extractant. The extract was concentrated under
reduced pressure into a small amount, into which petroleum ether
was added to precipitate 2.60 g of the title compound (44%) as a
colorless crystal, Mp 157-159.degree. C.
Example 3
Preparation of
1,3-dihydroxy-2-(4'-hydroxyphenyl)-4,4,5,5-tetramethylimidizolidine
[0091] 1.22 g (10 mmol) p-hydroxy benzaldehyde and 1.48 g (10 mmol)
2,3-dimethyl-2,3-dihydroxyaminobutane were dissolved in 10 mL
methanol and stirred at RT for 8 h until the starting material spot
disappeared as shown by TLC. Upon vacuum filtration, 1.29 g (51%)
title compound was obtained as a colorless crystal. ELMS (m/z) 252
[M].sup.+. .sup.1H-NMR (DMSO-d.sub.6) .delta. (ppm)=1.03 (s, 6H),
1.05 (s, 6H), 4.39 (s, 1H), 6.70 (d, J=6.9 Hz, 2H), 7.23 (d, J=6.9
Hz, 2H), 7.63 (s, 1H), 7.85 (s, 2H).
Example 4
Preparation of
1,3-dihydroxy-2-(4'-hydroxyphenyl)-4,4,5,5-tetramethylimidazoline
[0092] 504 mg (2 mmol)
1,3-dihydroxy-2-(4'-hydroxyphenyl)-4,4,5,5-imidizolidine were
dissolved in 30 mL methanol followed by addition of 3 g PbO.sub.2,
and stirred at RT for 40 min until the starting material spot
disappeared as shown by TLC. After removal of solids by vacuum
filtration, the filtrate was distilled until dry under reduced
pressure at RT, and the residue was purified by column
chromatography (with chloroform as the eluent) to afford 260 mg
(52%) of the title compound as blue solid. Mp 134-135.degree. C.,
ELMS (m/z) 249 [M].sup.+. IR (KBr) 3250, 1610, 1500, 1490, 840.
Example 5
Preparation of 1,3-dioxo-2-(4'-(oxyacetate ethyl
ester)-phenyl)-4,4,5,5-tetramethylimidazoline
[0093] 250 mg (1 mmol)
1,3-dihydroxy-2-(4'-hydroxyphenyl)-4,4,5,5-tetramethylimidazoline,
0.32 mL ethyl bromoacetate and 32 mg NaH were dissolved in 5 mL
anhydrous THF. The mixture was stirred at 60.degree. C. for 5 h
until the starting material spot disappeared as shown by TLC. After
filtration under reduced pressure at RT, the filtrate was
concentrated under reduced pressure till dry, and the residue was
purified by column chromatography (ethyl acetate:petroleum
ether=1:5) to give 300 mg (90%) of the target compound, MP
107-109.degree. C.
Example 6
Preparation of
1,3-dioxo-2-(4'-oxyacetoxy-phenyl)-4,4,5,5-tetramethylimidazoline
(TMMZ)
[0094] 7 drops of a NaOH (2N) aqueous solution was added into a
solution of 33 mg (0.1 mmol) 1,3-dioxo-2-(4'-(oxyacetate ethyl
ester)-phenyl)-4,4,5,5-tetramethylimidazoline in 3 mL methanol,
followed by stirring at RT for 30 min until the starting material
spot disappeared as shown by TLC. The reaction mixture was
concentrated under reduced pressure, and the residue was diluted by
addition of 2 mL saturated saline, adjusted to pH 6 with 2N HCl,
and then extracted 3 times with ethyl acetate (3 ml.times.3). The
layers of ethyl acetate phase were combined and dried over
anhydrous sodium sulfate and then filtered. The filtrate was
concentrated under reduced pressure at RT till dry to afford 30 mg
(99%) of the title compound as blue crystal. Mp 155-157.degree. C.
ELMS (m/z) 307 [M].sup.+.
Coupling the Imidazolines Having NO Free Radical Scavenging
Activity with a Linking Arm:
1,3-dioxo-2-[(4'-oxyacetyl-Lys-OMe)phenyl]-4,4,5,5-tetramethylimidazoline
Example 7
Preparation of
1,3-dioxo-2-[(4'-oxyacetyl-N.sup..omega.-Boc-Lys-OMe)phenyl]-4,4,5,5-tetr-
amethylimidazoline
[0095] A solution of 307 mg (1 mmol)
1,3-dioxo-2-(4'-oxyacetyl-phenyl)-4,4,5,5-tetramethylimidazoline in
30 ml anhydrous THF was stirred on an ice bath, into which 250 mg
(1.2 mmol) DCC and 135 mg (1 mmol) HOBt were added and stirred on
an ice bath for 10 min. Then a solution prepared with 300 mg (1
mmol) HCl.Lys(Boc)-Ome, 122 mg (1 mmol) N-methylmorpholine and 6 mL
anhydrous THF was added thereto, and the reaction mixture was
reacted at RT for 24 h. TLC (ethyl acetate:petroleum ether=2:1)
showed disappearance of HCl.Lys(Boc)-Ome. The reaction mixture was
concentrated under reduced pressure till dry, the residue was
dissolved in ethyl acetate and insoluble material was removed by
filtration. The filtrate was sequentially washed with a saturated
aqueous solution of sodium bicarbonate and with a saturated aqueous
solution of NaCl, the separated ethyl acetate phase was dried over
anhydrous sodium sulfate and filtered, and the filtrate was then
concentrated under reduce pressure at 37.degree. C. (these
operations were hereinafter referred to as a generic "routine
procedure"). The residue was purified by column chromatography
(ethyl acetate:petroleum ether=2:1) to give 433 mg (65%) of the
title compound as blue solid. ESI-MS(m/z) 550 [M+H].sup.+.
Example 8
Preparation of
1,3-dioxo-2-[(4'-oxyacetyl-Lys-OMe)phenyl]-4,4,5,5-tetramethylimidazoline
[0096] 625 mg (1 mmol)
1,3-dioxo-2-[(4'-oxyacetyl-W-Boc-Lys-OMe)phenyl]-4,4,5,5-tetramethylimida-
zoline was dissolved in 15 mL anhydrous hydrogen chloride-ethyl
acetate (4N) and stirred at RT for 3 h until the starting material
spot disappeared as shown by TLC (CHCl.sub.3:MeOH, 20:1). The
reaction mixture was then subjected to the routine procedure. The
residue was crystallized in anhydrous ethyl ether to afford the
title compound.
Preparation of the Peptide Having Thrombolytic Activity: Properly
Protected ARPAK
Example 9
Preparation of Boc-Ala-Lys(Z)-OBzl
[0097] 473 mg (2.5 mmol) Boc-Ala was dissolved in 10 ml anhydrous
THF. A solution prepared with 338 mg (2.5 mmol) HOBt, 619 mg (3
mmol) DCC and 10 mL anhydrous THF was added thereto on an ice bath.
The reaction mixture was stirred on the ice bath for 20 min before
a solution prepared with 936 mg (2.3 mmol) HCl.Lys(Z)-Obzl, 232 mg
(2.3 mmol) N-methyl morpholine and 6 mL anhydrous THF was added
thereto. The resultant reaction mixture reacted at RT for 24 h
until HCl.Lys(Z)-Obzl disappeared as shown by TLC
(CHCl.sub.3:MeOH=30:1). The reaction mixture was subjected to the
routine procedure to give 1.204 g (97%) of the title compound as
colorless solid. Mp 88-90.degree. C. [.alpha.].sub.D.sup.20=-29.2
(c=0.1, MeOH). ESI-MS(m/z) 565 [M+Na].sup.+.
Example 10
Preparation of HCl.Ala-Lys(Z)-OBzl
[0098] 1.354 g (2.5 mmol) Boc-Ala-Lys(Z)-Obzl was dissolved in
approximately 10 ml solution of anhydrous hydrogen chloride in
ethyl acetate (4N) and stirred at RT for 3 h until the starting
material spot disappeared as shown by TLC (CHCl.sub.3:MeOH, 30:1).
The reaction mixture solution was concentrated under reduced
pressure at RT, and the residue was dissolved in ethyl acetate and
then concentrated at RT; the above process was repeated for several
times until all free hydrogen chloride was removed (these
operations are hereinafter referred to as a "routine procedure").
The residue was crystallized in anhydrous ethyl ether to afford the
title compound which was directly used in the reaction of the next
step.
Example 11
Preparation of Boc-Pro-Ala-Lys(Z)-OBzl
[0099] 538 mg (2.5 mmol) Boc-Pro was dissolved in an appropriate
amount of anhydrous THF followed by addition of 338 mg (2.5 mmol)
HOBt and 619 mg (3 mmol) DCC in anhydrous THF on an ice bath, and
then reacted for 20 min. To this solution, a solution prepared with
1.099 g (2.3 mmol) HCl.Ala-Lys(Z)-Obzl and 232 mg (2.3 mmol)
N-methyl morpholine in 10 mL anhydrous THF was added, and the
reaction was carried out at RT for 24 h. TLC (CHCl.sub.3:MeOH,
20:1) showed disappearance of the starting material spot. The
reaction compounds were subjected to the routine procedure to
afford 2.847 g (98%) title compound, Mp 82-83.degree. C.
[.alpha.].sub.D.sup.20=-46.4 (c=0.11, MeOH). ESI-MS(m/z) 661
[M+Na].sup.+.
Example 12
Preparation of HCl.Pro-Ala-Lys(Z)-OBzl
[0100] 1.596 g (2.5 mmol) Boc-Pro-Ala-Lys(Z)-Obzl was dissolved in
a 15 mL solution of anhydrous hydrogen chloride in ethyl acetate
(4N) and stirred at RT for 3 h until the starting material spot
disappeared as shown by TLC (CHCl.sub.3:MeOH, 20:1). The reaction
mixture was subjected to the routine procedure, and the residue was
crystallized in anhydrous ethyl ether to give the title compound
which was directly used in the reaction of the next step.
Example 13
Preparation of Boc-Arg(NO.sub.2)-Pro-Ala-Lys(Z)-OBzl
[0101] On an ice bath, a solution of 798 mg (2.5 mmol)
Boc-Arg(NO.sub.2), 338 mg (2.5 mmol) HOBt, 619 mg (3 mmol) DCC in
10 mL anhydrous THF was stirred for 20 min, into which a solution
prepared with 1.322 g (2.3 mmol) HCl.Pro-Ala-Lys(Z)-Obzl and 232 mg
(2.3 mmol) N-methylmorpholine in 5 mL anhydrous THF was added and
the reaction was carried out at RT for 24 hours until the starting
material spot disappeared as shown by TLC (CHCl.sub.3:MeOH, 20:1).
The routine procedure was carried out to give 1.642 g (85%) of the
title compound. Mp 84-85.degree. C. [.alpha.].sub.D.sup.20=-65.0
(c=0.13, MeOH). ESI-MS (m/z) 864 [M+Na].sup.+.
Example 14
Preparation of HCl.Arg(NO.sub.2)-Pro-Ala-Lys(Z)-OBzl
[0102] 2.099 g (2.5 mmol) Boc-Arg(NO.sub.2)-Pro-Ala-Lys(Z)-OBzl was
dissolved in a 20 mL solution of anhydrous hydrogen chloride in
ethyl acetate (4N) and stirred at RT for 3 h until the starting
material spot disappeared as shown by TLC (CHCl.sub.3:MeOH, 20:1).
The reaction mixture was subjected to the routine procedure, and
the residue was crystallized in anhydrous ethyl ether to give the
title compound which was directly used in the reaction of the next
step.
Example 15
Preparation of Boc-Ala-Arg(NO.sub.2)-Pro-Ala-Lys(Z)-OBzl
[0103] On an ice bath, a solution of 473 mg (2.5 mmol) Boc-Ala, 338
mg (2.5 mmol) HOBt, 619 mg (3 mmol) DCC in 10 mL anhydrous THF was
stirred for 20 min, into which a solution prepared with 1.785 g
(2.3 mmol) HCl.Arg(NO.sub.2)-Pro-Ala-Lys(Z)-Obzl, 232 mg (2.3 mmol)
N-methylmorpholine in 5 mL anhydrous THF was added and the reaction
was carried out for 24 hours to give 1.802 g (86%) of the title
compound. Mp 87-89.degree. C. [.alpha.].sub.D.sup.20=-63.9 (c=0.12,
MeOH). ESI-MS (m/e) 934 [M+Na].sup.+.
Example 16
Preparation of Boc-Ala-Arg(NO.sub.2)-Pro-Ala-Lys(Z)
[0104] 921 mg (1 mmol) Boc-Ala-Arg(NO.sub.2)-Pro-Ala-Lys(Z)-OBzl
was dissolved in 3 mL methanol, into which a NaOH aqueous solution
(2N) was added on an ice bath and stirred at RT for 30 min. With pH
maintained at 12, the reaction was stirred on the ice bath for 10
min until the starting material spot disappeared as shown by TLC.
pH was adjusted to 7 with 2N HCl, and the reaction liquid was
concentrated under reduced pressure. The residue was diluted with 2
mL saturated saline and adjusted to pH 2 with 2N HCl, and then
extracted 3 times with ethyl acetate (5 mL.times.3). The layers of
ethyl acetate phase were combined and dried over anhydrous sodium
sulfate, and concentrated under reduced pressure at RT to afford
767 mg (80%) of title compound as colorless solid. ELMS (m/z) 830
[M-H].sup.-.
Preparation of Thrombus Targeting/Anti-Thrombus Peptide: Properly
Protected RGDS, RGDV, RGDF
Example 17
Preparation of Boc-Asp(OBzl)-Ser(Bzl)-OBzl
[0105] On an ice bath, a solution of 808 mg (2.5 mmol)
Boc-Asp(OBzl), 338 mg (2.5 mmol) HOBt, 619 mg (3 mmol) DCC in 10 mL
anhydrous THF was stirred and reacted for 20 min, and then a
solution prepared with 740 mg (2.3 mmol) HCl.Ser(Bzl)-Obzl, 232 mg
(2.3 mmol) N-methylmorpholine in 5 mL anhydrous THF was added
thereto and reacted at RT for 24 h until the starting material
disappeared as shown by TLC (CHCl.sub.3:MeOH, 20:1). The reaction
compounds were subjected to the routine procedure to afford 1.29 g
(95%) of the title compound as colorless oily matter. ESI-MS(m/z)
591 [M+H].sup.+.
Example 18
Preparation of HCl.Asp(OBzl)-Ser(Bzl)-OBzl
[0106] 1.477 g (2.5 mmol) Boc-Asp(OBzl)-Ser(Bzl)-Obzl was dissolved
in a 15 mL solution of anhydrous hydrogen chloride in ethyl acetate
(4N) and stirred at RT for 3 h until the starting material
disappeared as shown by TLC (CHCl.sub.3:MeOH, 20:1). The reaction
mixture was subjected to the routine procedure, and the residue was
crystallized in anhydrous ethyl ether to give the title compound
which was directly used in the reaction of the next step.
Example 19
Preparation of Boc-Gly-Asp(OBzl)-Ser(Bzl)-OBzl
[0107] On an ice bath, a solution of 438 mg (2.5 mmol) Boc-Gly, 338
mg (2.5 mmol) HOBt, 619 mg (3 mmol) DCC in 10 mL anhydrous THF was
stirred for 20 min, and then a solution prepared with 1.212 g (2.3
mmol) HCl.Asp(OBzl)-Ser(Bzl)-Obzl and 232 mg (2.3 mmol)
N-methylmorpholine in 5 mL anhydrous THF was added thereto and
reacted at RT for 24 h until the starting material disappeared as
shown by TLC (CHCl.sub.3:MeOH, 20:1). The reaction mixture was
subjected to the routine procedure to afford 1.461 g (98%) of the
title compound as colorless solid. Mp 53-55.degree. C.
[.alpha.].sub.D.sup.20=-23.7 (c=0.13, MeOH). ESI-MS (m/z) 649
[M+H].sup.+.
Example 20
Preparation of HCl.Gly-Asp(OBzl)-Ser(Bzl)-OBzl
[0108] 1.619 g (2.5 mmol) Boc-Gly-Asp(OBzl)-Ser(Bzl)-Obzl was
dissolved in a 15 mL solution of anhydrous hydrogen chloride in
ethyl acetate (4N) and stirred at RT for 3 h until the starting
material spot disappeared as shown by TLC (CHCl.sub.3:MeOH, 20:1).
The reaction mixture was subjected to the routine procedure, and
the residue was crystallized in anhydrous ethyl ether to give the
title compound which was directly used in the reaction of the next
step.
Example 21
Preparation of Boc-Arg(NO.sub.2)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl
[0109] On an ice bath, a solution of 798 mg (2.5 mmol)
Boc-Arg(NO.sub.2), 338 mg (2.5 mmol) HOBt, 619 mg (3 mmol) DCC in
10 mL anhydrous THF was stirred for 20 min, and then a solution
prepared with 1.343 g (2.3 mmol) HCl.Gly-Asp(OBzl)-Ser(Bzl)-Obzl
and 232 mg (2.3 mmol) N-methylmorpholine in 5 mL anhydrous THF was
added thereto and reacted at RT for 24 h until the starting
material disappeared as shown by TLC (CHCl.sub.3:MeOH, 20:1). After
the routine procedure, 1.66 g (85%) of the title compound was
obtained as colorless solid. Mp 74-75.degree. C.
[.alpha.].sub.D.sup.20=-26.2 (c=0.12, MeOH). ESI-MS(m/z) 872
[M+Na].sup.+.
Example 22
Preparation of HCl.Arg(NO.sub.2)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl
[0110] A mixture of 2.122 g (2.5 mmol)
Boc-Arg(NO.sub.2)-Gly-Asp(OBzl)-Ser(Bzl)-Obzl and a 20 mL solution
of hydrogen chloride in ethyl acetate (4N) was stirred at RT for 3
h until the starting material spot disappeared as shown by TLC
(CHCl.sub.3: MeOH, 20:1). The reaction mixture was subjected to the
routine procedure, the residue was crystallized in anhydrous ethyl
ether to afford the title compound.
Example 23
Preparation of Boc-Asp(OBzl)-Val-OBzl
[0111] On an ice bath, a solution of 808 mg (2.5 mmol)
Boc-Asp(OBzl), 338 mg (2.5 mmol) HOBt, 619 mg (3 mmol) DCC in 10 ml
anhydrous THF was stirred for 20 min, and then a solution prepared
with 558 mg (2.3 mmol) HCl.Val-Obzl and 232 mg (2.3 mmol)
N-methylmorpholine in 5 mL anhydrous THF was added thereto and
reacted at RT for 24 h until the starting material disappeared as
shown by TLC (CHCl.sub.3:MeOH, 20:1). The reaction mixture was
subjected to the routine procedure to afford 1.129 g (96%) of the
title compound as colorless oily liquid. ESI-MS(m/z) 512
[M+H].sup.+.
Example 24
Preparation of HCl.Asp(OBzl)-Val-OBzl
[0112] 1.278 g (2.5 mmol) Boc-Asp(OBzl)-Val-OBzl was dissolved in a
15 mL solution of anhydrous hydrogen chloride in ethyl acetate (4N)
and stirred at RT for 3 h until the starting material spot
disappeared as shown by TLC (CHCl.sub.3:MeOH, 20:1). The reaction
mixture was subjected to the routine procedure, and the residue was
crystallized in anhydrous ethyl ether to give the title compound
which was directly used in the reaction of the next step.
Example 25
Preparation of Boc-Gly-Asp(OBzl)-Val-OBzl
[0113] On an ice bath, a solution of 438 mg (2.5 mmol) Boc-Gly, 338
mg (2.5 mmol) HOBt, 619 mg (3 mmol) DCC in 10 ml anhydrous THF was
stirred for 20 min, and then a solution prepared with 1.03 g (2.3
mmol) HCl.Asp(OBzl)-Val-Obzl and 232 mg (2.3 mmol)
N-methylmorpholine in 5 mL anhydrous THF was added thereto and
reacted at RT for 24 h until the starting material disappeared as
shown by TLC (CHCl.sub.3:MeOH, 20:1). The reaction mixture was
subjected to the routine procedure to afford 1.242 g (95%) of the
title compound as colorless solid. Mp 66-68.degree. C.
[.alpha.].sub.D.sup.20=-43.8 (c=0.11, MeOH). ESI-MS(m/z) 592
[M+Na].sup.+.
Example 26
Preparation of HCl.Gly-Asp(OBzl)-Val-OBzl
[0114] 1.421 g (2.5 mmol) Boc-Gly-Asp(OBzl)-Val-OBzl was dissolved
in a 15 mL solution of anhydrous hydrogen chloride in ethyl acetate
(4N) and stirred at RT for 3 h until the starting material spot
disappeared as shown by TLC (CHCl.sub.3:MeOH, 20:1). The reaction
mixture was subjected to the routine procedure, and the residue was
crystallized in anhydrous ethyl ether to give the title compound
which was directly used in the reaction of the next step.
Example 27
Preparation of Boc-Arg(NO.sub.2)-Gly-Asp(OBzl)-Val-OBzl
[0115] On an ice bath, a solution of 798 mg (2.5 mmol)
Boc-Arg(NO.sub.2), 338 mg (2.5 mmol) HOBt, 619 mg (3 mmol) DCC in
10 ml anhydrous THF was stirred for 20 min, and then a solution
prepared with 1.162 g (2.3 mmol) HCl.Gly-Asp(OBzl)-Val-Obzl and 232
mg (2.3 mmol) N-methylmorpholine in 5 mL anhydrous THF was added
thereto and reacted at RT for 24 h until the starting material
disappeared as shown by TLC (CHCl.sub.3:MeOH, 20:1). The reaction
mixture was subjected to the routine procedure to afford 1.523 g
(86%) of the title compound as colorless solid. Mp 107-109.degree.
C. [.alpha.].sub.D.sup.20=-38.0 (c=0.12, MeOH). ESI-MS(m/z) 793
[M+Na].sup.+.
Example 28
Preparation of HCl.Gly-Asp(OBzl)-Val-OBzl
[0116] 1.925 g (2.5 mmol) Boc-Arg(NO.sub.2)-Gly-Asp(OBzl)-Val-OBzl
was dissolved in a 20 mL solution of anhydrous hydrogen chloride in
ethyl acetate (4N) and stirred at RT for 3 h until the starting
material spot disappeared as shown by TLC (CHCl.sub.3:MeOH, 20:1).
The reaction mixture was subjected to the routine procedure, and
the residue was crystallized in anhydrous ethyl ether to give the
title compound.
Example 29
Preparation of Boc-Asp(OBzl)-Phe-OBzl
[0117] On an ice bath, a solution of 808 mg (2.5 mmol)
Boc-Asp(OBzl), 338 mg (2.5 mmol) HOBt, 619 mg (3 mmol) DCC in 10 ml
anhydrous THF was stirred for 20 min, and then a solution prepared
with 668 mg (2.3 mmol) HCl.Phe-Obzl and 232 mg (2.3 mmol)
N-methylmorpholine in 5 mL anhydrous THF was added thereto and
reacted at RT for 24 h until the starting material disappeared as
shown by TLC (CHCl.sub.3:MeOH, 20:1). After the routine procedure,
1.222 g (95%) of the title compound was obtained as colorless
solid. Mp 79-80.degree. C. [.alpha.].sub.D.sup.20=-24.2 (c=0.13,
MeOH), ESI-MS(m/z) 561 [M+H].sup.+.
Example 30
Preparation of HCl.Asp(OBzl)-Phe-OBzl
[0118] 1.398 g (2.5 mmol) Boc-Asp(OBzl)-Phe-OBzl was dissolved in a
15 mL solution of anhydrous hydrogen chloride in ethyl acetate (4N)
and stirred at RT for 3 h until the starting material spot
disappeared as shown by TLC (CHCl.sub.3:MeOH, 20:1). The reaction
mixture was subjected to the routine procedure, and the residue was
crystallized in anhydrous ethyl ether to give the title compound
which was directly used in the reaction of the next step.
Example 31
Preparation of Boc-Gly-Asp(OBzl)-Phe-OBzl
[0119] On an ice bath, a solution of 438 mg (2.5 mmol) Boc-Gly, 338
mg (2.5 mmol) HOBt, 619 mg (3 mmol) DCC in anhydrous THF was
stirred for 20 min, and then a solution prepared with 1.141 g (2.3
mmol) HCl.Asp(OBzl)-Phe-Obzl and 232 mg (2.3 mmol)
N-methylmorpholine in 5 mL anhydrous THF was added thereto and
reacted at RT for 24 h until the starting material disappeared as
shown by TLC (CHCl.sub.3:MeOH, 20:1). The reaction mixture was
subjected to the routine procedure to afford 1.29 g (91%) of the
title compound as colorless solid. Mp 70-71.degree. C.
[.alpha.].sub.D.sup.20=-22.5 (c=0.14, MeOH). ESI-MS(m/z) 640
[M+Na].sup.+.
Example 32
Preparation of HCl.Gly-Asp(OBzl)-Phe-OBzl
[0120] 1.541 g (2.5 mmol) Boc-Gly-Asp(OBzl)-Phe-OBzl was dissolved
in a 15 mL solution of anhydrous hydrogen chloride in ethyl acetate
(4N) and stirred at RT for 3 h until the starting material spot
disappeared as shown by TLC (CHCl.sub.3:MeOH, 20:1). The reaction
mixture was subjected to the routine procedure, and the residue was
crystallized in anhydrous ethyl ether to give the title compound
which was directly used in the reaction of the next step.
Example 33
Boc-Arg(NO.sub.2)-Gly-Asp(OBzl)-Phe-OBzl
[0121] On an ice bath, a solution of 798 mg (2.5 mmol)
Boc-Arg(NO.sub.2), 338 mg (2.5 mmol) HOBt, 619 mg (3 mmol) DCC in
10 ml anhydrous THF was stirred for 20 min, and then a solution
prepared with 1.272 g (2.3 mmol) HCl.Gly-Asp(OBzl)-Phe-Obzl and 232
mg (2.3 mmol) N-methylmorpholine in 5 mL anhydrous THF was added
thereto and reacted at RT for 24 h until the starting material
disappeared as shown by TLC (CHCl.sub.3:MeOH, 20:1). The reaction
mixture was subjected to the routine procedure to afford 1.637 g
(87%) of the title compound as colorless solid. Mp 77-79.degree. C.
[.alpha.].sub.D.sup.20=-22.6 (c=0.09, MeOH). ESI-MS(m/z) 841
[M+Na].sup.+.
Example 34
Preparation of HCl.Arg(NO.sub.2)-Gly-Asp(OBzl)-Phe-OBzl
[0122] 2.045 g (2.5 mmol) Boc-Arg(NO.sub.2)-Gly-Asp(OBzl)-Phe-OBzl
was dissolved in a 15 mL solution of anhydrous hydrogen chloride in
ethyl acetate (4N) and stirred at RT for 3 h until the starting
material spot disappeared as shown by TLC (CHCl.sub.3:MeOH, 20:1).
The reaction mixture was subjected to the routine procedure, and
the residue was crystallized in anhydrous ethyl ether to give the
title compound.
Preparation of Ternary Conjugates of ARPAK/Imidazoline/RGD
(Compounds of General Formula I-1-1): Ia, Ib, Ic
Example 35
Preparation of
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Ala-Arg(NO.sub.2)-Pro-Ala-L-
ys(Z)]-Lys-OMe}phenyl}-4,4,5,5-tetramethylimidazoline
[0123] On an ice bath, a solution of 821 mg (1 mmol)
Boc-Ala-Arg(NO.sub.2)-Pro-Ala-Lys(Z), 135 mg (1 mmol) HOBt and 250
mg (1 mmol) DCC in 10 mL anhydrous THF was stirred for 20 min, and
then a solution prepared with 480 mg (1 mmol)
1,3-dioxo-2-[(4'-oxyacetyl-Lys-OMe)phenyl]-4,4,5,5-tetramethylimidazoline
and 100 mg (1 mmol) N-methylmorpholine in 5 mL anhydrous THF was
added thereto and reacted at RT for 24 h until the starting
material disappeared as shown by TLC (CHCl.sub.3:MeOH, 40:1). The
reaction mixture was subjected to the routine procedure to afford
925 mg (83%) of the title compound as blue solid. Mp
179-182.degree. C. [.alpha.].sub.D.sup.20=-34.3 (c=0.14, MeOH),
ESI-MS(m/z) 1275 [M+Na].sup.+. IR (KBr) 3319, 2935, 1658, 1531,
1448, 1363, 1254, 1168, 1053, 835, 749, 540 cm.sup.-1.
Example 36
Preparation of
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Ala-Arg(NO.sub.2)-Pro-Ala-L-
ys(Z)]-Lys}phenyl}-4,4,5,5-tetramethylimidazoline
[0124] On an ice bath, 1260 mg (1 mmol)
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Ala-Arg(NO.sub.2)-Pro-Ala-L-
ys(Z)]-Lys-OMe}phenyl}-4,4,5,5-tetramethylimidazoline was dissolved
in 3 ml methanol followed by addition of a NaOH aqueous solution
(2N), and then stirred at RT for 30 min. With pH maintained at 12,
the reaction was stirred on the ice bath for 10 min until the
starting material disappeared as shown by TLC. With pH adjusted to
7 with 2N HCl, the reaction liquid was concentrated under reduced
pressure, and the residue was diluted in 2 mL saturated saline,
adjusted to pH 2 with 2N HCl, and then extracted 3 times with ethyl
acetate (5 mL.times.3). The combined ethyl acetate phase was dried
over anhydrous sodium sulfate and filtered, and the filtrate was
concentrated under reduced pressure at RT to afford 945 mg (82%) of
the title compound as blue solid. ELMS (m/z) 1238 [M-H].sup.-.
Example 37
Preparation of
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Ala-Arg(NO.sub.2)-Pro-Ala-L-
ys(Z)]-Lys-Arg-(NO.sub.2)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl}phenyl}-4,4,5,5-tetr-
amethylimidazoline
[0125] On an ice bath, a solution of 618 mg (0.5 mmol)
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Ala-Arg(NO.sub.2)-Pro-Ala-L-
ys (Z)]-Lys}phenyl}-4,4,5,5-tetramethylimidazoline, 69 mg (0.5
mmol) HOBt and 126 mg (0.6 mmol) DCC in 20 mL anhydrous THF was
stirred for 20 min, and then a solution prepared with 442 mg (0.5
mmol) HCl.Arg(NO.sub.2)-Gly-Asp(OBzl)-Ser(Bzl)-Obzl and 50 mg (0.5
mmol) N-methylmorpholine in 5 mL anhydrous THF was added thereto
and reacted at RT for 24 h until the starting material disappeared
as shown by TLC (CHCl.sub.3:MeOH, 20:1). The reaction mixture was
subjected to the routine procedure to afford 300 mg (31%) of the
title compound as blue solid. Mp 138-140.degree. C.
[.alpha.].sub.D.sup.20=-39.4 (c=0.13, MeOH). ESI-MS(m/z) 1991
[M+H].sup.+. IR(KBr) 3309, 2936, 1656, 1531, 1449, 1363, 1256, 836,
743, 697, 601 cm.sup.-1.
Example 38
Preparation of
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Ala-Arg(NO.sub.2)-Pro-Ala-L-
ys(Z)]-Lys-Arg-(NO.sub.2)-Gly-Asp(OBzl)-Val-OBzl}phenyl}-4,4,5,5-tetrameth-
ylimidazoline
[0126] On an ice bath, a solution of 618 mg (0.5 mmol)
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Ala-Arg(NO.sub.2)-Pro-Ala-L-
ys(Z)]-Lys}phenyl}-4,4,5,5-tetramethylimidazoline, 69 mg (0.5 mmol)
HOBt and 126 mg (0.6 mmol) DCC in 20 mL anhydrous THF was stirred
for 20 min, and then a solution prepared with 421 mg (0.5 mmol)
HCl.Arg(NO.sub.2)-Gly-Asp(OBzl)-Val-Obzl and 50 mg (0.5 mmol)
N-methylmorpholine in 5 mL anhydrous THF was added thereto and
reacted at RT for 24 h until the starting material disappeared as
shown by TLC (CHCl.sub.3:MeOH, 20:1). The reaction mixture was
subjected to the routine procedure to afford 389 mg (36%) of the
title compound as blue solid. Mp 117-120.degree. C.
[.alpha.].sub.D.sup.20=-14.8 (c=0.01, MeOH). ESI-MS(m/z) 1913
[M+H].sup.+. IR(KBr) 3312, 2937, 1655, 1530, 1448, 1362, 1257, 835,
744, 697, 592 cm.sup.-1.
Example 39
Preparation of
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Ala-Arg(NO.sub.2)-Pro-Ala-L-
ys(Z)]-Lys-Arg-(NO.sub.2)-Gly-Asp(OBzl)-Phe-OBzl}phenyl}-4,4,5,5-tetrameth-
ylimidazoline
[0127] On an ice bath, a solution of 618 mg (0.5 mmol)
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Ala-Arg(NO.sub.2)-Pro-Ala-L-
ys(Z)]-Lys}phenyl}-4,4,5,5-tetramethylimidazoline, 69 mg (0.5 mmol)
HOBt and 126 mg (0.6 mmol) DCC in 20 mL anhydrous THF was stirred
for 20 min, and then a solution prepared with 445 mg (0.5 mmol)
HCl.Arg(NO.sub.2)-Gly-Asp(OBzl)-Phe-Obzl and 50 mg (0.5 mmol)
N-methylmorpholine in 5 mL anhydrous THF was added thereto and
reacted at RT for 24 h until the starting material disappeared as
shown by TLC (CHCl.sub.3:MeOH, 20:1). The reaction mixture was
subjected to the routine procedure to afford 320 mg (36%) of the
title compound as blue solid. Mp 115-118.degree. C.
[.alpha.].sub.D.sup.20=-21.5 (c=0.16, MeOH). ESI-MS (m/z) 1961
[M+H].sup.+. IR(KBr) 3316, 2936, 1654, 1529, 1448, 1362, 1256,
1169, 742, 698, 593 cm.sup.-1.
Example 40
Preparation of
1,3-dioxo-2-{4'-oxyacetyl-[N.sup..omega.-(Ala-Arg-Pro-Ala-Lys)-Lys-Arg-Gl-
y-Asp-Ser]phenyl}-4,4,5,5-tetramethylimidazoline (Ia)
[0128] On an ice bath, 199 mg (0.1 mmol)
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Ala-Arg(NO.sub.2)--Pro-Ala--
Lys
(Z)]-Lys-Arg-(NO.sub.2)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl}phenyl}-4,4,5,5-te-
tramethylimidazoline was mixed with 6 mL trifluoroacetic acid and
1.5 mL trifluoromethanesulfonic acid, and stirred for 1 h until the
starting material disappeared as shown by TLC (CHCl.sub.3:MeOH,
1:1). The reaction mixture was concentrated under reduced pressure,
and the residue was repeatedly washed with anhydrous ethyl ether
and concentrated under reduced pressure. The residue was dissolved
in water, adjusted to pH=8 with 25% ammonia water, desalted with
Sephadex G10, and then purified on a C18 column. The collected
fractions were lyophilized to afford 109 mg (85%) of the title
compound as blue solid. Mp 134-135.degree. C.
[.alpha.].sub.D.sup.20=-39.7 (c=0.12, MeOH). FT-MS(m/z) 1374.7290
[M+H].sup.+, 2748.4580 [2M+H].sup.+, 4122.1870 [3M+H].sup.+,
5495.9160 [4M+H].sup.+. g=2.00779. IR(KBr) 3346, 3180, 2920, 1665,
1537, 1449, 1252, 1179, 1030, 837, 801, 720, 639, 518
cm.sup.-1.
Example 41
Preparation of
1,3-dioxo-2-{4'-oxyacetyl-[N.sup..omega.-(Ala-Arg-Pro-Ala-Lys)-Lys-Arg-Gl-
y-Asp-Val]phenyl}-4,4,5,5-tetramethylimidazoline (Ib)
[0129] On an ice bath, 190 mg (0.1 mmol)
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Ala-Arg(NO.sub.2)-Pro-Ala-L-
ys(Z)]-Lys-Arg-(NO.sub.2)-Gly-Asp(OBzl)-Val-OBzl}phenyl}-4,4,5,5-tetrameth-
ylimidazoline was mixed with 6 mL trifluoroacetic acid and 1.5 mL
trifluoromethanesulfonic acid, and stirred for 1 h until the
starting material disappeared as shown by TLC (CHCl.sub.3:MeOH,
1:1). The reaction mixture was concentrated under reduced pressure,
and the residue was repeatedly washed with anhydrous ethyl ether
and concentrated under reduced pressure. The residue was dissolved
in water, adjusted to pH 8 with 25% ammonia water, desalted with
Sephadex G10, and then purified on a C18 column. The collected
fractions were lyophilized to afford 96 mg (82%) of the title
compound as blue solid. Mp 143-144.degree. C.
[.alpha.].sub.D.sup.20=-31.8 (c=0.01, MeOH). FT-MS(m/z) 1386.7654
[M+H].sup.+, 2772.5308, [2M+H].sup.+, 4158.2962 [3M+H].sup.+,
5544.0616 [4M+H].sup.+. g=2.00779. IR(KBr) 3349, 2942, 1659, 1539,
1394, 1250, 1030, 639 cm.sup.-1.
Example 42
Preparation of
1,3-dioxo-2-{4'-oxyacetyl-[N.sup..omega.-(Ala-Arg-Pro-Ala-Lys)-Lys-Arg-Gl-
y-Asp-Phe]phenyl}-4,4,5,5-tetramethylimidazoline (Ic)
[0130] On an ice bath, 194 mg (0.1 mmol)
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Ala-Arg(NO.sub.2)-Pro-Ala-L-
ys(Z)]-Lys-Arg-(NO.sub.2)-Gly-Asp(OBzl)-Phe-OBzl}phenyl}-4,4,5,5-tetrameth-
ylimidazoline was mixed with 6 mL trifluoroacetic acid and 1.5 mL
trifluoromethanesulfonic acid, and stirred for 1 h until the
starting material disappeared as shown by TLC (CHCl.sub.3:MeOH,
1:1). The reaction mixture was concentrated under reduced pressure,
and the residue was repeatedly washed with anhydrous ethyl ether
and concentrated under reduced pressure. The residue was dissolved
in water, adjusted to pH 8 with 25% ammonia water, desalted with
Sephadex G10, and then purified on a C18 column. The collected
fractions were lyophilized to afford 106 mg (81%) of the title
compound as blue solid. Mp 96-97.degree. C.
[.alpha.].sub.D.sup.20=-44.4 (c=0.15, MeOH). FT-MS (m/z) ESI-MS
(m/z) 1444.7654 [M+H].sup.+, ESI-MS(m/z) 2888.5308 [2M+H].sup.+,
4332.2962 [3M+H].sup.+, 5776.0616 [4M+H].sup.+. g=2.00789. IR(KBr)
3363, 1665, 1538, 1448, 1256, 1173, 1031, 640, 577, 518
cm.sup.-1.
Preparation of the Peptide Having Thrombolytic Activity: Properly
Protected GRPAK
Example 43
Preparation of Boc-Gly-Arg(NO.sub.2)-Pro-Ala-Lys(Z)-OBzl
[0131] On an ice bath, a solution of 438 mg (2.5 mmol) Boc-Gly, 338
mg (2.5 mmol) HOBt, 619 mg (3 mmol) DCC in 10 ml anhydrous THF was
stirred for 20 min, and then a solution prepared with 1.785 g (2.3
mmol) HCl.Arg(NO.sub.2)-Pro-Ala-Lys(Z)-Obzl and 232 mg (2.3 mmol)
N-methylmorpholine in 5 mL anhydrous THF was added thereto and
reacted at RT for 24 h to give 1.857 g (90%) of the title compound.
Mp 85-87.degree. C. [.alpha.].sub.D.sup.20=-38.5 (c=0.11, MeOH).
ESI-MS (m/e) 920 [M+Na].sup.+.
Example 44
Preparation of Boc-Gly-Arg(NO.sub.2)-Pro-Ala-Lys(Z)
[0132] 907 mg (1 mmol) Boc-Gly-Arg(NO.sub.2)-Pro-Ala-Lys(Z)-OBzl
was dissolved in 3 ml methanol followed by addition of a NaOH
aqueous solution (2N) on an ice bath, and then stirred at RT for 30
min. With pH maintained at 12, the reaction was stirred on the ice
bath for 10 min until the starting material disappeared as shown by
TLC. With pH adjusted to 7 with 2N HCl, the reaction liquid was
concentrated under reduced pressure, and the residue was diluted in
2 mL saturated saline, adjusted to pH 2 with 2N HCl, and then
extracted 3 times with ethyl acetate (5 mL.times.3). The combined
ethyl acetate phase was dried over anhydrous sodium sulfate, and
then concentrated under reduced pressure at RT to afford 785 mg
(82%) of the title compound as colorless solid. ELMS (m/z) 816
[M-H].sup.-.
Preparation of Ternary Conjugates of GRPAK/Imidazoline/RGD
(Compounds of General formula I-1-2): Id, Ie, If
Example 45
Preparation of
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Gly-Arg(NO.sub.2)-Pro-Ala-L-
ys(Z)]-Lys-OMe}phenyl}-4,4,5,5-tetramethylimidazoline
[0133] On an ice bath, a solution of 817 mg (1 mmol)
Boc-Gly-Arg(NO.sub.2)-Pro-Ala-Lys(Z), 135 mg (1 mmol) HOBt and 250
mg (1 mmol) DCC in 10 mL anhydrous THF was stirred for 20 min, and
then a solution prepared with 480 mg (1 mmol)
1,3-dioxo-2-[(4'-oxyacetyl-Lys-OMe)phenyl]-4,4,5,5-tetramethylimidazoline
and 100 mg (1 mmol) N-methylmorpholine in 5 mL anhydrous THF was
added thereto and reacted at RT for 24 h until the starting
material disappeared as shown by TLC (CHCl.sub.3:MeOH, 40:1). The
reaction mixture was subjected to the routine procedure to afford
680 mg (52%) of the title compound as blue solid. Mp 79-82.degree.
C. [.alpha.].sub.D.sup.20=-12.3 (c=0.14, MeOH), ESI-MS(m/z) 1261
[M+Na].sup.+. IR (KBr) 3319, 2935, 1658, 1531, 1448, 1363, 1254,
1168, 1053, 835, 749, 540 cm.sup.-1.
Example 46
Preparation of
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Gly-Arg(NO.sub.2)-Pro-Ala-L-
ys(Z)]-Lys}phenyl}-4,4,5,5-tetramethylimidazoline
[0134] On an ice bath, 1260 mg (1 mmol)
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Gly-Arg(NO.sub.2)-Pro-Ala-L-
ys(Z)]-Lys-OMe}phenyl}-4,4,5,5-tetramethylimidazoline was dissolved
in 3 ml methanol followed by addition of a NaOH aqueous solution
(2N), and then stirred at RT for 30 min. With pH maintained at 12,
the reaction was stirred on the ice bath for 10 min until the
starting material disappeared as shown by TLC. With pH adjusted to
7 with 2N HCl, the reaction liquid was concentrated under reduced
pressure, and the residue was diluted in 2 mL saturated saline,
adjusted to pH 2 with 2N HCl, and then extracted 3 times with ethyl
acetate (5 mL.times.3). The combined ethyl acetate phase was dried
over anhydrous sodium sulfate and filtered, and the filtrate was
concentrated under reduced pressure at RT to afford 945 mg (82%) of
the title compound as colorless solid. ELMS (m/z) 1223
[M-H].sup.-.
Example 47
Preparation of
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Gly-Arg(NO.sub.2)-Pro-Ala-L-
ys(Z)]-Lys-Arg-(NO.sub.2)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl}phenyl}-4,4,5,5-tetr-
amethylimidazoline
[0135] On an ice bath, a solution of 611 mg (0.5 mmol)
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Gly-Arg(NO.sub.2)-Pro-Ala-L-
ys(Z)]-Lys}phenyl}-4,4,5,5-tetramethylimidazoline, 69 mg (0.5 mmol)
HOBt and 126 mg (0.6 mmol) DCC in 20 mL anhydrous THF was stirred
for 20 min, and then a solution prepared with 442 mg (0.5 mmol)
HCl.Arg(NO.sub.2)-Gly-Asp(OBzl)-Ser(Bzl)-Obzl and 50 mg (0.5 mmol)
N-methylmorpholine in 5 mL anhydrous THF was added thereto and
reacted at RT for 24 h until the starting material disappeared as
shown by TLC (CHCl.sub.3:MeOH, 20:1). The reaction mixture was
subjected to the routine procedure to afford 500 mg (48%) of the
title compound as blue solid. Mp 127-129.degree. C.
[.alpha.].sub.D.sup.20=-49.4 (c=0.13, MeOH). ESI-MS(m/z) 1956
[M+H]'. IR(KBr) 3306, 2936, 1652, 1531, 1449, 1362, 1255, 1166,
742, 697, 592 cm.sup.-1.
Example 48
Preparation of
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Gly-Arg(NO.sub.2)-Pro-Ala-L-
ys(Z)]-Lys-Arg-(NO.sub.2)-Gly-Asp(OBzl)-Val-OBzl}phenyl}-4,4,5,5-tetrameth-
ylimidazoline
[0136] On an ice bath, a solution of 611 mg (0.5 mmol)
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Gly-Arg(NO.sub.2)-Pro-Ala-L-
ys (Z)]-Lys}phenyl}-4,4,5,5-tetramethylimidazoline, 69 mg (0.5
mmol) HOBt and 126 mg (0.6 mmol) DCC in 20 mL anhydrous THF was
stirred for 20 min, and then a solution prepared with 421 mg (0.5
mmol) HCl.Arg(NO.sub.2)-Gly-Asp(OBzl)-Val-Obzl, 50 mg (0.5 mmol)
N-methylmorpholine in 5 mL anhydrous THF was added thereto and
reacted at RT for 24 h until the starting material disappeared as
shown by TLC (CHCl.sub.3:MeOH, 20:1). The reaction mixture was
subjected to the routine procedure to afford 392 mg (35%) of the
title compound as blue solid. Mp 147-150.degree. C.
[.alpha.].sub.D.sup.20=-34.6 (c=0.16, MeOH). ESI-MS(m/z) 1899
[M+Na].sup.+. IR(KBr) 3311, 3068, 2937, 1661, 1531, 1451, 1395,
1254, 1163, 839, 743, 697, 596 cm.sup.-1.
Example 49
Preparation of
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Gly-Arg(NO.sub.2)-Pro-Ala-L-
ys(Z)]-Lys-Arg-(NO.sub.2)-Gly-Asp(OBzl)-Phe-OBzl}phenyl}-4,4,5,5-tetrameth-
ylimidazoline
[0137] On an ice bath, a solution of 611 mg (0.5 mmol)
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Gly-Arg(NO.sub.2)-Pro-Ala-L-
ys(Z)]-Lys}phenyl}-4,4,5,5-tetramethylimidazoline, 69 mg (0.5 mmol)
HOBt and 126 mg (0.6 mmol) DCC in 20 mL anhydrous THF was stirred
for 20 min, and then a solution prepared with 445 mg (0.5 mmol)
HCl.Arg(NO.sub.2)-Gly-Asp(OBzl)-Phe-Obzl and 50 mg (0.5 mmol)
N-methylmorpholine in 5 mL anhydrous THF was added thereto and
reacted at RT for 24 h until the starting material disappeared as
shown by TLC (CHCl.sub.3:MeOH, 20:1). The reaction mixture was
subjected to the routine procedure to afford 336 mg (31%) of the
title compound as blue solid. Mp 125-128.degree. C.
[.alpha.].sub.D.sup.20=-31.3 (c=0.18, MeOH). ESI-MS (m/z) 1925
[M+H].sup.+. IR(KBr) 3315, 2935, 1657, 1529, 1448, 1361, 1257,
1173, 834, 742, 698, 541 cm.sup.-1.
Example 50
Preparation of
1,3-dioxo-2-{4'-oxyacetyl-[N.sup..omega.-(Gly-Arg-Pro-Ala-Lys)-Lys-Arg-Gl-
y-Asp-Ser]phenyl}-4,4,5,5-tetramethylimidazoline (Id)
[0138] On an ice bath, 195 mg (0.1 mmol)
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Gly-Arg(NO.sub.2)-Pro-Ala-L-
ys(Z)]-Lys-Arg-(NO.sub.2)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl}phenyl}-4,4,5,5-tetr-
amethylimidazoline was mixed with 6 mL trifluoroacetic acid and 1.5
mL trifluoromethanesulfonic acid, and stirred for 1 h until the
starting material disappeared as shown by TLC (CHCl.sub.3:MeOH,
1:1). The reaction mixture was concentrated under reduced pressure,
and the residue was repeatedly washed with anhydrous ethyl ether
and concentrated under reduced pressure. The residue was dissolved
in water, adjusted to pH 8 with 25% ammonia water, desalted with
Sephadex G10, and then purified on a C18 column. The collected
fractions were lyophilized to afford 102 mg (82%) of the title
compound as blue solid. Mp 142-145.degree. C.
[.alpha.].sub.D.sup.20=29.7 (c=0.14, MeOH). FT-MS(m/z) 1360.7133
[M+H].sup.+, 2720.4266 [2M+H].sup.+, 4080.1399 [3M+H].sup.+,
5439.8532 [4M+H].sup.+. g=2.00779. IR(KBr) 3348, 3180, 2940, 1670,
1539, 1447, 1199, 1134, 1034, 836, 801, 721, 638 cm.sup.-1.
Example 51
Preparation of
1,3-dioxo-2-{4'-oxyacetyl-[N.sup..omega.-(Gly-Arg-Pro-Ala-Lys)-Lys-Arg-Gl-
y-Asp-Val]phenyl}-4,4,5,5-tetramethylimidazoline (Ie)
[0139] On an ice bath, 190 mg (0.1 mmol)
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Gly-Arg(NO.sub.2)--Pro-Ala--
Lys (Z)]-Lys-Arg-(NO.sub.2)-Gly-Asp
(OBzl)-Val-OBzl}phenyl}-4,4,5,5-tetramethylimidazoline was mixed
with 6 mL trifluoroacetic acid and 1.5 mL trifluoromethanesulfonic
acid, and stirred for 1 h until the starting material disappeared
as shown by TLC (CHCl.sub.3:MeOH, 1:1). The reaction mixture was
concentrated under reduced pressure, and the residue was repeatedly
washed with anhydrous ethyl ether and concentrated under reduced
pressure. The residue was dissolved in water, adjusted to pH 8 with
25% ammonia water, desalted with Sephadex G10, and then purified on
a C18 column. The collected fractions were lyophilized to afford 99
mg (84%) of the title compound as blue solid. Mp 147-149.degree. C.
[.alpha.].sub.D.sup.20=31.1 (c=0.17, MeOH). FT-MS(m/z) 1372.7497
[M+H].sup.+, 2744.4994 [2M+H].sup.+, 4116.2491 [3M+H].sup.+,
5487.9988 [4M+H].sup.+. g=2.00779. IR(KBr) 3338, 2960, 1662, 1539,
1451, 1392, 1251, 1170, 1030, 639, 519 cm.sup.-1.
Example 52
Preparation of
1,3-dioxo-2-{4'-oxyacetyl-[N.sup..omega.-(Gly-Arg-Pro-Ala-Lys)-Lys-Arg-Gl-
y-Asp-Phe]phenyl}-4,4,5,5-tetramethylimidazoline (If)
[0140] On an ice bath, 192 mg (0.1 mmol)
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Gly-Arg(NO.sub.2)-Pro-Ala-L-
ys(Z)]-Lys-Arg-(NO.sub.2)-Gly-Asp(OBzl)-Phe-OBzl}phenyl}-4,4,5,5-tetrameth-
ylimidazoline was mixed with 6 mL trifluoroacetic acid and 1.5 mL
trifluoromethanesulfonic acid, and stirred for 1 h until the
starting material disappeared as shown by TLC (CHCl.sub.3:MeOH,
1:1). The reaction mixture was concentrated under reduced pressure,
and the residue was repeatedly washed with anhydrous ethyl ether
and concentrated under reduced pressure. The residue was dissolved
in water, adjusted to pH 8 with 25% ammonia water, desalted with
Sephadex G10, and then purified on a C18 column. The collected
fractions were lyophilized to afford 106 mg (81%) of the title
compound as blue solid. Mp 84-85.degree. C.
[.alpha.].sub.D.sup.20=-54.1 (c=0.15, MeOH). FT-MS (m/z) 1420.7497
[M+H]', 2840.4994 [2M+H].sup.+, ESI-MS FT-MS(m/z) 1420.7497
[M+H].sup.+, 2840.4994 [2M+H].sup.+, 4260.2491 [3M+H].sup.+,
5679.9976 [4M+H].sup.+. g=2.00789. IR(KBr) 3344, 3080, 2930, 1666,
1535, 1392, 1250, 1181, 1030, 835, 800, 719, 638 cm.sup.-1.
Preparation of the Peptide Having Thrombolytic Activity: Properly
Protected RPAK
Example 53
Preparation of Boc-Arg(NO.sub.2)-Pro-Ala-Lys(Z)
[0141] On an ice bath, 850 mg (1 mmol)
Boc-Arg(NO.sub.2)-Pro-Ala-Lys(Z)-OBzl was dissolved in 3 ml
methanol followed by addition of a NaOH aqueous solution (2N), and
then stirred at RT for 30 min. With pH maintained at 12, the
reaction was stirred on the ice bath for 10 min until the starting
material disappeared as shown by TLC. With pH adjusted to 7 with 2N
HCl, the reaction liquid was concentrated under reduced pressure,
and the residue was diluted in 2 mL saturated saline, adjusted to
pH 2 with 2N HCl, and then extracted 3 times with ethyl acetate (5
mL.times.3). The combined ethyl acetate phase was dried over
anhydrous sodium sulfate, and filtered, then the filtrate is
concentrated under reduced pressure at RT to afford 742 mg (92%) of
the title compound as colorless solid. EI-MS (m/z) 849
[M-H].sup.-.
Preparation of Ternary Conjugates of RPAK/Imidazoline/RGD
(Compounds of General formula I-1-3): Ig, Ih, Ii
Example 54
Preparation of
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Arg(NO.sub.2)-Pro-Ala-Lys(Z-
)]-Lys-OMe}phenyl}-4,4,5,5-tetramethylimidazoline
[0142] On an ice bath, a solution of 760 mg (1 mmol)
Boc-Arg(NO.sub.2)-Pro-Ala-Lys(Z), 135 mg (1 mmol) HOBt and 250 mg
(1 mmol) DCC in 10 mL anhydrous THF was stirred for 20 min, and
then a solution prepared with 480 mg (1 mmol)
1,3-dioxo-2-[(4'-oxyacetyl-Lys-OMe)phenyl]-4,4,5,5-tetramethylimidazoline
and 100 mg (1 mmol) N-methylmorpholine in 5 mL anhydrous THF was
added thereto and reacted at RT for 24 h until the starting
material disappeared as shown by TLC (CHCl.sub.3:MeOH, 40:1). The
reaction mixture was subjected to the routine procedure to afford
920 mg (83%) of the title compound as blue solid. Mp 72-76.degree.
C. [.alpha.].sub.D.sup.20=-32.7 (c=0.13, MeOH), ESI-MS(m/z) 1204
[M+Na].sup.+. IR (KBr) 3317, 2937, 1658, 1531, 1447, 1362, 1254,
1168, 1055, 835, 746, 697, 541, 460 cm.sup.-1.
Example 55
Preparation of
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Arg(NO.sub.2)-Pro-Ala-Lys(Z-
)]-Lys}phenyl}-4,4,5,5-tetramethylimidazoline
[0143] On an ice bath, 1200 mg (1 mmol)
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Arg(NO.sub.2)-Pro-Ala-Lys(Z-
)]-Lys-OMe}phenyl}-4,4,5,5-tetramethylimidazoline was dissolved in
3 ml methanol followed by addition of a NaOH aqueous solution (2N),
and then stirred at RT for 30 min. With pH maintained at 12, the
reaction was stirred on the ice bath for 10 min until the starting
material disappeared as shown by TLC. With pH adjusted to 7 with 2N
HCl, the reaction liquid was concentrated under reduced pressure,
and the residue was diluted in 2 mL saturated saline, adjusted to
pH 2 with 2N HCl, and then extracted 3 times with ethyl acetate (5
mL.times.3). The combined ethyl acetate phase was dried over
anhydrous sodium sulfate and filtered, and the filtrate was
concentrated under reduced pressure at RT to afford 899 mg (80%) of
the title compound as blue solid. EI-MS (m/z) 1116 [M-H].sup.-.
Example 56
Preparation of
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Arg(NO.sub.2)-Pro-Ala-Lys(Z-
)]-Lys-Arg-(NO.sub.2)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl}phenyl}-4,4,5,5-tetramet-
hylimidazoline
[0144] On an ice bath, a solution of 583 mg (0.5 mmol)
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Arg(NO.sub.2)-Pro-Ala-Lys(Z-
)]-Lys}phenyl}-4,4,5,5-tetramethylimidazoline, 69 mg (0.5 mmol)
HOBt and 126 mg (0.6 mmol) DCC in 20 mL anhydrous THF was stirred
for 20 min, and then a solution prepared with 442 mg (0.5 mmol)
HCl.Arg(NO.sub.2)-Gly-Asp(OBzl)-Ser(Bzl)-Obzl and 50 mg (0.5 mmol)
N-methylmorpholine in 5 mL anhydrous THF was added thereto and
reacted at RT for 24 h until the starting material disappeared as
shown by TLC (CHCl.sub.3:MeOH, 20:1). The reaction mixture was
subjected to the routine procedure to afford 421 mg (40%) of the
title compound as blue solid. Mp 77-79.degree. C.
[.alpha.].sub.D.sup.20=-45.4 (c=0.15, MeOH). ESI-MS(m/z) 1897
[M+H].sup.+. IR(KBr) 3319, 2934, 1658, 1530, 1449, 1361, 1256, 834,
741, 698, 542 cm.sup.-1.
Example 57
Preparation of
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Arg(NO.sub.2)-Pro-Ala-Lys(Z-
)]-Lys-Arg-(NO.sub.2)-Gly-Asp(OBzl)-Val-OBzl}phenyl}-4,4,5,5-tetramethylim-
idazoline
[0145] On an ice bath, a solution of 583 mg (0.5 mmol)
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Arg(NO.sub.2)-Pro-Ala-Lys
(Z)]-Lys}phenyl}-4,4,5,5-tetramethylimidazoline, 69 mg (0.5 mmol)
HOBt and 126 mg (0.6 mmol) DCC in 20 mL anhydrous THF was stirred
for 20 min, and then a solution prepared with 421 mg (0.5 mmol)
HCl.Arg(NO.sub.2)-Gly-Asp(OBzl)-Val-Obzl and 50 mg (0.5 mmol)
N-methylmorpholine in 5 mL anhydrous THF was added thereto and
reacted at RT for 24 h until the starting material disappeared as
shown by TLC (CHCl.sub.3:MeOH, 20:1). The reaction mixture was
subjected to the routine procedure to afford 472 mg (42%) of the
title compound as blue solid. Mp 107-109.degree. C.
[.alpha.].sub.D.sup.20=-28.8 (c=0.11, MeOH). ESI-MS(m/z) 1820
[M+H].sup.+. IR(KBr) 3314, 2938, 1658, 1531, 1448, 1362, 1258, 742,
698, 594 cm.sup.-1.
Example 58
Preparation of
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Arg(NO.sub.2)-Pro-Ala-Lys(Z-
)]-Lys-Arg-(NO.sub.2)-Gly-Asp(OBzl)-Phe-OBzl}phenyl}-4,4,5,5-tetramethylim-
idazoline
[0146] On an ice bath, a solution of 583 mg (0.5 mmol)
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Arg(NO.sub.2)-Pro-Ala-Lys(Z-
)]-Lys}phenyl}-4,4,5,5-tetramethylimidazoline, 69 mg (0.5 mmol)
HOBt and 126 mg (0.6 mmol) DCC in 20 mL anhydrous THF was stirred
for 20 min, and then a solution prepared with 445 mg (0.5 mmol)
HCl.Arg(NO.sub.2)-Gly-Asp(OBzl)-Phe-Obzl and 50 mg (0.5 mmol)
N-methylmorpholine in 5 mL anhydrous THF was added thereto and
reacted at RT for 24 h until the starting material disappeared as
shown by TLC (CHCl.sub.3:MeOH, 20:1). The reaction mixture was
subjected to the routine procedure to afford 420 mg (47%) of the
title compound as blue solid. Mp 141-144.degree. C.
[.alpha.].sub.D.sup.20=-35.7 (c=0.12, MeOH). ESI-MS(m/z) 1867
[M+H].sup.+. IR(KBr) 3319, 2936, 1656, 1529, 1448, 1362, 1257,
1169, 834, 743, 698, 541 cm.sup.-1.
Example 59
Preparation of
1,3-dioxo-2-{4'-oxyacetyl-[N.sup..omega.-(Arg-Pro-Ala-Lys)-Lys-Arg-Gly-As-
p-Ser]phenyl}-4,4,5,5-tetramethylimidazoline (Ig)
[0147] On an ice bath, 170 mg (0.1 mmol)
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Arg(NO.sub.2)-Pro-Ala-Lys
(Z)]-Lys-Arg-(NO.sub.2)-Gly-Asp
(OBzl)-Ser(Bzl)-OBzl}phenyl}-4,4,5,5-tetramethylimidazoline was
mixed with 6 mL trifluoroacetic acid and 1.5 mL
trifluoromethanesulfonic acid, and stirred for 1 h until the
starting material disappeared as shown by TLC (CHCl.sub.3:MeOH,
1:1). The reaction mixture was concentrated under reduced pressure,
and the residue was repeatedly washed with anhydrous ethyl ether
and concentrated under reduced pressure. The residue was dissolved
in water, adjusted to pH 8 with 25% ammonia water, desalted with
Sephadex G10, and then purified on a C18 column. The collected
fractions were lyophilized to afford 102 mg (82%) of the title
compound as blue solid. Mp 148-150.degree. C.
[.alpha.]D.sup.20=-22.4 (c=0.14, MeOH). FT-MS(m/z) 1303.6919
[M+H].sup.+, 2606.3838, [2M+H].sup.+, 3909.0757 [3M+H].sup.+,
5211.7676 [4M+H].sup.+. g=2.00779. IR(KBr) 3344, 3080, 2930, 1666,
1535, 1392, 1250, 1181, 1030, 835, 800, 719, 638.
Example 60
Preparation of
1,3-dioxo-2-{4'-oxyacetyl-[N.sup..omega.-(Arg-Pro-Ala-Lys)-Lys-Arg-Gly-As-
p-Val]phenyl}-4,4,5,5-tetramethylimidazoline (Ih)
[0148] On an ice bath, 182 mg (0.1 mmol)
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Arg(NO.sub.2)-Pro-Ala-Lys
(Z)]-Lys-Arg-(NO.sub.2)-Gly-Asp(OBzl)-Val-OBzl}phenyl}-4,4,5,5-tetramethy-
limidazoline was mixed with 6 mL trifluoroacetic acid and 1.5 mL
trifluoromethanesulfonic acid, and stirred for 1 h until the
starting material disappeared as shown by TLC (CHCl.sub.3:MeOH,
1:1). The reaction mixture was concentrated under reduced pressure,
and the residue was repeatedly washed with anhydrous ethyl ether
and concentrated under reduced pressure. The residue was dissolved
in water, adjusted to pH 8 with 25% ammonia water, desalted with
Sephadex G10, and then purified on a C18 column. The collected
fractions were lyophilized to afford 99 mg (84%) of the title
compound as blue solid. Mp 137-139.degree. C.
[.alpha.].sub.D.sup.20=-34.3 (c=0.18, MeOH). FT-MS(m/z) ESI-MS(m/z)
1315.7282 [M+H].sup.+, 2630.4564 [2M+H].sup.+, 3945.1846
[3M+H].sup.+, 5259.9128 [4M+H].sup.+. g=2.00779. IR(KBr) 3329,
2953, 1665, 1533, 1391, 1198, 1134, 834, 801, 720, 599
cm.sup.-1.
Example 61
Preparation of
1,3-dioxo-2-{4'-oxyacetyl-[N.sup..omega.-(Arg-Pro-Ala-Lys)-Lys-Arg-Gly-As-
p-Phe]phenyl}-4,4,5,5-tetramethylimidazoline (Ii)
[0149] On an ice bath, 187 mg (0.1 mmol)
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Arg(NO.sub.2)-Pro-Ala-Lys
(Z)]-Lys-Arg-(NO.sub.2)-Gly-Asp(OBzl)-Phe-OBzl}phenyl}-4,4,5,5-tetramethy-
limidazoline was mixed with 6 mL trifluoroacetic acid and 1.5 mL
trifluoromethanesulfonic acid, and stirred for 1 h until the
starting material disappeared as shown by TLC (CHCl.sub.3:MeOH,
1:1). The reaction mixture was concentrated under reduced pressure,
and the residue was repeatedly washed with anhydrous ethyl ether
and concentrated under reduced pressure. The residue was dissolved
in water, adjusted to pH 8 with 25% ammonia water, desalted with
Sephadex G10, and then purified on a C18 column. The collected
fractions were lyophilized to afford 96 mg (81%) of the title
compound as blue solid. Mp 99-100.degree. C.
[.alpha.].sub.D.sup.20=-24.7 (c=0.14, MeOH). FT-MS (m/z) 1363.7282
[M+H].sup.+, 2726.4564 [2M+H].sup.+, 4089.1846 [3M+H].sup.+,
5451.9128 [4M+H].sup.+. g=2.00789. IR(KBr) 3322, 3060, 2928, 1661,
1530, 1391, 1303, 1247, 641 cm.sup.-1.
Preparation of the Peptide Having Thrombolytic Activity: Properly
Protected Pak
Example 62
Preparation of Boc-Pro-Ala-Lys(Z)
[0150] On an ice bath, 638 mg (1 mmol) Boc-Pro-Ala-Lys(Z)-OBzl was
dissolved in 3 ml methanol followed by addition of a NaOH aqueous
solution (2N), and then stirred at RT for 30 min. With pH
maintained at 12, the reaction was stirred on the ice bath for 10
min until the starting material disappeared as shown by TLC. With
pH adjusted to 7 with 2N HCl, the reaction liquid was concentrated
under reduced pressure, and the residue was diluted in 2 mL
saturated saline, adjusted to pH 2 with 2N HCl, and then extracted
3 times with ethyl acetate (5 mL.times.3). The combined ethyl
acetate phase was dried over anhydrous sodium sulfate and filtered,
and the filtrate was then concentrated under reduced pressure at RT
to afford 509 mg (91.6%) of the title compound as colorless solid.
ELMS (m/z) 547 [M-H].sup.-.
Preparation of Ternary Conjugates of PAK/Imidazoline/RGD (Compounds
of General Formula I-1-4): Ij, Ik, Il
Example 63
Preparation of
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Pro-Ala-Lys(Z)]-Lys-OMe}phe-
nyl}-4,4,5,5-tetramethylimidazoline
[0151] On an ice bath, a solution of 548 mg (1 mmol)
Boc-Pro-Ala-Lys(Z), 135 mg (1 mmol) HOBt and 250 mg (1 mmol) DCC in
10 mL anhydrous THF was stirred for 20 min, and then a solution
prepared with 480 mg (1 mmol)
1,3-dioxo-2-[(4'-oxyacetyl-Lys-OMe)phenyl]-4,4,5,5-tetramethylimidazoline
and 100 mg (1 mmol) N-methylmorpholine in 5 mL anhydrous THF was
added thereto and reacted at RT for 24 h until the starting
material disappeared as shown by TLC (CHCl.sub.3:MeOH, 40:1). The
reaction mixture was subjected to the routine procedure to afford
876 mg (87%) of the title compound as blue solid. Mp 77-80.degree.
C. [.alpha.].sub.D.sup.20=-12.6 (c=0.16, MeOH). ESI-MS(m/z) 1003
[M+Na].sup.+. IR (KBr): 3315, 3069, 2937, 1671, 1531, 1449, 1394,
1364, 1302, 1167, 1132, 1054, 836, 743, 698, 596, 541, 458
cm.sup.-1.
Example 64
Preparation of
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Pro-Ala-Lys(Z)]-Lys}phenyl}-
-4,4,5,5-tetramethylimidazoline
[0152] On an ice bath, 980 mg (1 mmol)
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Pro-Ala-Lys(Z)]-Lys-OMe}phe-
nyl}-4,4,5,5-tetramethylimidazoline was dissolved in 3 ml methanol
followed by addition of a NaOH aqueous solution (2N), and then
stirred at RT for 30 min. With pH maintained at 12, the reaction
was stirred on the ice bath for 10 min until the starting material
disappeared as shown by TLC. With pH adjusted to 7 with 2N HCl, the
reaction liquid was concentrated under reduced pressure, and the
residue was diluted in 2 mL saturated saline, adjusted to pH 2 with
2N HCl, and then extracted 3 times with ethyl acetate (5
mL.times.3). The combined ethyl acetate phase was dried over
anhydrous sodium sulfate and filtered, and the filtrate was
concentrated under reduced pressure at RT to afford 867 mg (80%) of
the title compound as blue solid. ELMS (m/z) 965 [M-H].sup.-.
Example 65
Preparation of
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Pro-Ala-Lys(Z)]-Lys-Arg-(NO-
.sub.2)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl}phenyl}-4,4,5,5-tetramethylimidazoline
[0153] On an ice bath, a solution of 483 mg (0.5 mmol)
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Pro-Ala-Lys(Z)]-Lys}phenyl}-
-4,4,5,5-tetramethylimidazoline, 69 mg (0.5 mmol) HOBt and 126 mg
(0.6 mmol) DCC in 20 mL anhydrous THF was stirred for 20 min, and
then a solution prepared with 442 mg (0.5 mmol)
HCl.Arg(NO.sub.2)-Gly-Asp(OBzl)-Ser(Bzl)-Obzl and 50 mg (0.5 mmol)
N-methylmorpholine in 5 mL anhydrous THF was added thereto and
reacted at RT for 24 h until the starting material disappeared as
shown by TLC (CHCl.sub.3:MeOH, 20:1). The reaction mixture was
subjected to the routine procedure to afford 421 mg (42%) of the
title compound as blue solid. Mp 97-100.degree. C.
[.alpha.].sub.D.sup.20=-42.5 (c=0.14, MeOH). ESI-MS(m/z) 1697
[M+H]'. IR(KBr) 3298, 3070, 2935, 2869, 1642, 1534, 1450, 1369,
1253, 741, 697, 596 cm.sup.-1.
Example 66
Preparation of
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Pro-Ala-Lys(Z)]-Lys-Arg-(NO-
.sub.2)-Gly-Asp(OBzl)-Val-OBzl}phenyl}-4,4,5,5-tetramethylimidazoline
[0154] On an ice bath, a solution of 483 mg (0.5 mmol)
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Pro-Ala-Lys(Z)]-Lys}phenyl}-
-4,4,5,5-tetramethylimidazoline, 69 mg (0.5 mmol) HOBt and 126 mg
(0.6 mmol) DCC in 20 mL anhydrous THF was stirred for 20 min, and
then a solution prepared with 432 mg (0.5 mmol)
HCl.Arg(NO.sub.2)-Gly-Asp(OBzl)-Val-Obzl and 50 mg (0.5 mmol)
N-methylmorpholine in 5 mL anhydrous THF was added thereto and
reacted at RT for 24 h until the starting material disappeared as
shown by TLC (CHCl.sub.3:MeOH, 20:1). The reaction mixture was
subjected to the routine procedure to afford 357 mg (37%) of the
title compound as blue solid. Mp 117-120.degree. C.
[.alpha.].sub.D.sup.20=-22.3 (c=0.17, MeOH). ESI-MS(m/z) 1620
[M+H].sup.+. IR(KBr) 3303, 3072, 2935, 1644, 1533, 1451, 1394,
1364, 1255, 1167, 745, 697, 597 cm.sup.-1.
Example 67
Preparation of
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Pro-Ala-Lys(Z)]-Lys-Arg-(NO-
.sub.2)-Gly-Asp(OBzl)-Phe-OBzl}phenyl}-4,4,5,5-tetramethylimidazoline
[0155] On an ice bath, a solution of 483 mg (0.5 mmol)
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Pro-Ala-Lys(Z)]-Lys}phenyl}-
-4,4,5,5-tetramethylimidazoline, 69 mg (0.5 mmol) HOBt and 126 mg
(0.6 mmol) DCC in 20 mL anhydrous THF was stirred for 20 min, and
then a solution prepared with 439 mg (0.5 mmol)
HCl.Arg(NO.sub.2)-Gly-Asp(OBzl)-Phe-Obzl and 50 mg (0.5 mmol)
N-methylmorpholine in 5 mL anhydrous THF was added thereto and
reacted at RT for 24 h until the starting material disappeared as
shown by TLC (CHCl.sub.3:MeOH, 20:1). The reaction mixture was
subjected to the routine procedure to afford 472 mg (48%) of the
title compound as blue solid. Mp 111-114.degree. C.
[.alpha.].sub.D.sup.20=-15.3 (c=0.13, MeOH). ESI-MS(m/z) 1667
[M+H].sup.+. IR(KBr) 3296, 3071, 2935, 1641, 1534, 1394, 1253,
1170, 834, 745, 697, 594 cm.sup.-1.
Example 68
Preparation of
1,3-dioxo-2-{4'-oxyacetyl-[N.sup..omega.-(Pro-Ala-Lys)-Lys-Arg-Gly-Asp-Se-
r]phenyl}-4,4,5,5-tetramethylimidazoline (Ij)
[0156] On an ice bath, 169 mg (0.1 mmol)
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Pro-Ala-Lys(Z)]-Lys-Arg-(NO-
.sub.2)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl}phenyl}-4,4,5,5-tetramethylimidazoline
was mixed with 6 mL trifluoroacetic acid and 1.5 mL
trifluoromethanesulfonic acid, and stirred for 1 h until the
starting material disappeared as shown by TLC (CHCl.sub.3:MeOH,
1:1). The reaction mixture was concentrated under reduced pressure,
and the residue was repeatedly washed with anhydrous ethyl ether
and concentrated under reduced pressure. The residue was dissolved
in water, adjusted to pH 8 with 25% ammonia water, desalted with
Sephadex G10, and then purified on a C18 column. The collected
fractions were lyophilized to afford 98 mg (80%) of the title
compound as blue solid. Mp 127-128.degree. C.
[.alpha.].sub.D.sup.20=-22.4 (c=0.13, MeOH). FT-MS(m/z) 1147.5907
[M+H].sup.+, 2294.1814 [2M+H].sup.+, 3440.7721 [3M+H].sup.+,
4587.3628 [4M+H].sup.+. g=2.00779. IR(KBr) 3204, 1672, 1543, 1436,
1199, 1133, 837, 801, 722, 598 cm.sup.-1.
Example 69
Preparation of
1,3-dioxo-2-{4'-oxyacetyl-[N.sup..omega.-(Pro-Ala-Lys)-Lys-Arg-Gly-Asp-Va-
l]phenyl}-4,4,5,5-tetramethylimidazoline (Ik)
[0157] On an ice bath, 162 mg (0.1 mmol)
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Pro-Ala-Lys(Z)]-Lys-Arg-(NO-
.sub.2)-Gly-Asp(OBzl)-Val-OBzl}phenyl}-4,4,5,5-tetramethylimidazoline
was mixed with 6 mL trifluoroacetic acid and 1.5 mL
trifluoromethanesulfonic acid, and stirred for 1 h until the
starting material disappeared as shown by TLC (CHCl.sub.3:MeOH,
1:1). The reaction mixture was concentrated under reduced pressure,
and the residue was repeatedly washed with anhydrous ethyl ether
and concentrated under reduced pressure. The residue was dissolved
in water, adjusted to pH 8 with 25% ammonia water, desalted with
Sephadex G10, and then purified on a C18 column. The collected
fractions were lyophilized to afford 96 mg (81%) of the title
compound as blue solid. Mp 123-124.degree. C.
[.alpha.].sub.D.sup.20=-24.6 (c=0.13, MeOH). FT-MS(m/z) 1159.6271
[M+H].sup.+, 2318.2542 [2M+H].sup.+, 3476.8813 [3M+H].sup.+,
4635.5084 [4M+H].sup.+. g=2.00779. IR(KBr) 3388, 2959, 1666, 1540,
1494, 1198, 1134, 835, 801, 720, 598 cm.sup.-1.
Example 70
Preparation of
1,3-dioxo-2-{4'-oxyacetyl-[N.sup..omega.-(Pro-Ala-Lys)-Lys-Arg-Gly-Asp-Ph-
e]phenyl}-4,4,5,5-tetramethylimidazoline (Il)
[0158] On an ice bath, 169 mg (0.1 mmol)
1,3-dioxo-2-{4'-oxyacetyl-{N.sup..omega.-[Boc-Pro-Ala-Lys(Z)]-Lys-Arg-(NO-
.sub.2)-Gly-Asp(OBzl)-Phe-OBzl}phenyl}-4,4,5,5-tetramethylimidazoline
was mixed with 6 mL trifluoroacetic acid and 1.5 mL
trifluoromethanesulfonic acid, and stirred for 1 h until the
starting material disappeared as shown by TLC (CHCl.sub.3:MeOH,
1:1). The reaction mixture was concentrated under reduced pressure,
and the residue was repeatedly washed with anhydrous ethyl ether
and concentrated under reduced pressure. The residue was dissolved
in water, adjusted to pH 8 with 25% ammonia water, desalted with
Sephadex G10, and then purified on a C18 column. The collected
fractions were lyophilized to afford 96 mg (81%) of the title
compound as blue solid. Mp 153-154.degree. C.
[.alpha.].sub.D.sup.20=-12.6 (c=0.16, MeOH). FT-MS (m/z) 1207.6271
[M+H].sup.+, 2414.2542 [2M+H].sup.+, 3620.8813 [3M+H].sup.+,
4827.5084 [4M+H].sup.+. g=2.00789. IR(KBr) 3385, 2938, 1659, 1541,
1450, 1391, 1251, 1126, 963, 841, 599, 456 cm.sup.-1.
Experimental Example 1
Experiments on NO Radical Scavenging by Compounds Ia to Il of the
Present Invention
[0159] Male Wistar rats weighing 250 to 300 g were starved for 12 h
before operation with free access to drinking water, and sacrificed
by cervical dislocation. Thoracotomy was immediately carried out
and thoracic aorta was taken out, connective tissues attached
thereto were dissected, and vessels were cut into aorta rings with
a length of 3 to 5 mm and placed into a perfusion bath. The bath
contained 15 ml Krebs-Henseleit solution and was kept at a constant
temperature of 37.degree. C., into which 95% O.sub.2-5% CO.sub.2
gas was charged. The anchor to which the aorta rings were
immobilized was connected to a tension transducer, and vasomotion
curves were recorded on a dual-trace recorder at a paper speed of 1
mm/min. With the static tension adjusted to 1.0 g and 30 min of
equilibration, norepinephrine at a final concentration of
10.sup.-8M was dosed to allow the aorta to contract for
preexcitation. Norepinephrine was washed off, followed by 30 min of
equilibration, and norepinephrine was added into the bath to a
final concentration of 10.sup.-8 M. When the contraction tension
was steady at a plateau level, 20 .mu.l normal saline (blank), a 20
.mu.l solution of any one of compounds Ia to Il in normal saline
(at a final concentration of 5.times.10.sup.-6 M), or a 20 .mu.l
solution of NO free radical scavenger
(1,3-dioxo-2-(4'-oxyacetoxyl-phenyl)-4,4,5,5-tetramethylimidazoline,
TMMZ) in normal saline (at a final concentration of
5.times.10.sup.-6 M) was added into the bath. When stabilized, a 20
.mu.l acetylcholine in normal saline was added (at a final
concentration of 10.sup.-6 M). The NO scavenging ability of the
drugs was expressed as a percentage of inhibition of acetylcholine
induced vasodilation. The experimental results are shown in Table
1.
[0160] As shown in the experimental results, Ia to Il were able to
inhibit acetylcholine's vasodilating effect on the vessel pieces by
scavenging NO. As such, by linking a thrombolytic peptide ARPAK,
GRPAK, RPAK or PAK and a targeting peptide RGDS, RGDV or RGDF to a
free radical scavenger
(1,3-dioxo-2-(4'-oxyacetoxyl-phenyl)-4,4,5,5-tetramethylimidazoline,
TMMZ) via Lys, 9 compounds had substantially higher activity in
inhibition of acetylcholine-induced vasodilatation than TMMZ, 2
compounds had the same activity in inhibition of
acetylcholine-induced vasodilatation as TMMZ, and one compound was
less active in inhibition of acetylcholine-induced vasodilatation
than TMMZ. Among the 12 compounds under assessment, 4 compounds had
a percentage of inhibition higher than 30%, and these 4 compounds
were ranked by activity in inhibition of acetylcholine-induced
vasodilatation as Ie>Ih>Ib>If. This demonstrated that the
activity of the TMMZ moiety in scavenging NO free radicals was
generally improved by linking the thrombolytic peptide ARPAK,
GRPAK, RPAK or PAK and the targeting peptide RGDS, RGDV or RGDF to
the free radical scavenger TMMZ via Lys.
TABLE-US-00001 TABLE 1 Percentage of Ia to Il's inhibition of
vasodilatation induced by acetylcholine Percentage of inhibition
Compounds (Mean .+-. SD %) TMMZ 15.47 .+-. 2.20 .sup. Ia 22.82 .+-.
3.27 .sup.a Ib 35.32 .+-. 4.74 .sup.a Ic 21.78 .+-. 3.11 .sup.a Id
17.60 .+-. 2.75 .sup.b Ie 41.28 .+-. 3.27 .sup.a If 32.55 .+-. 2.55
.sup.a Ig 24.40 .+-. 3.60 .sup.a Ih 37.54 .+-. 1.84 .sup.a Ii 13.75
.+-. 2.07 .sup.b Ij 27.22 .+-. 2.68 .sup.a Ik 11.13 .+-. 2.92
.sup.c Il 22.62 .+-. 3.60 .sup.a n = 6; .sup.a p < 0.01 vs.
TMMZ; .sup.b p > 0.05 vs. TMMZ; .sup.c p < 0.05 vs. TMMZ
Experimental Example 2
Experiments on Euglobulin Clot Lysis by Compounds Ia to Il of the
Present Invention
[0161] Pig blood was taken and mixed with 3.8% sodium citrate in a
volume ratio of 9:1, immediately centrifuged at 3000 r/min for 10
min, and platelet-poor plasma was separated. 2 mL platelet-poor pig
plasma and 36 mL ultrapure water were added into a 50 mL centrifuge
tube. In each tube, 0.4 mL acetic acid (1%) was added and
thoroughly mixed, and the tube was placed in a 4.degree. C.
refrigerator for 10 min and then centrifuged at 3000 r/min for 10
min. The centrifuge tubes were inverted, and then the inner wall of
the tubes was dried using a filter paper after the liquid was
drained. The euglobuin pellets resulting from centrifugation was
freeze-dried for about 40 min and scratched out. About 35 mg
euglobuin was taken and dissolved in 7 ml borax buffer (pH 9.28).
The euglobuin were mostly dissolved after 1 h, into which 0.7 mL
CaCl.sub.2 solution (25 mM) was added, and immediately plated on a
10.times.10 cm glass plate with a thickness of about 1 mm. After
clot formation, 10 .mu.L normal saline, or 10 .mu.L of a solution
of one of compounds Ia to Il in normal saline (1 mM) or 10 .mu.L of
a urokinase solution in normal saline (0.8 mg/mL) was pipetted and
spotted onto the clot plate, with an interval between every two
drops more than 1.5 cm, and each sample was spotted 3 times. The
diameter of the clot lysis circle was measured after 4 h, and the
readings are listed in Table 2.
[0162] As shown in the experimental results, by linking a
thrombolytic peptide ARPAK, GRPAK, RPAK or PAK and a targeting
peptide RGDS, RGDV or RGDF to a free radical scavenger TMMZ via
Lys, all compounds exhibited substantial euglobulin clot lysis
activity.
TABLE-US-00002 TABLE 2 The diameter of euglobulin clot lysis after
4 h of Ia-Il treatment Compounds Diameter (Mean .+-. SD mm) Normal
saline .sup. 2.9 .+-. 0.6 .sup.a urokinase .sup. 10.7 .+-. 0.4
.sup.a Ia 4.0 .+-. 0.0 Ib 5.2 .+-. 0.3 Ic 3.8 .+-. 0.3 Id 5.2 .+-.
0.3 Ie 5.5 .+-. 0.3 If 4.5 .+-. 0.5 Ig 5.2 .+-. 0.3 Ih 4.2 .+-. 0.3
Ii 4.0 .+-. 0.0 Ij 4.0 .+-. 0.0 Ik 4.5 .+-. 0.5 Il 4.2 .+-. 0.3 n =
3; .sup.a p < 0.01, vs. Ia-l
Experimental Example 3
In Vitro Thrombolysis Experiments for Compounds Ia to Il of the
Present Invention
[0163] SD rats (male, 350 to 400 g) were anaesthetized by
intraperitoneal injection of a urethane solution at a dosage of
1200 mg/kg. The anaesthetized rats were fixed in a supine position,
and the right common carotid artery was dissected, clamped at the
proximal end with an arterial clip, and penetrated with a suture at
the proximal and distal ends, respectively. The suture at the
distal end is clipped tightly by a hemostatic clamp at the fur.
Cannulation was performed at the distal end, the artery clamp was
removed, and the total arterial blood was discharged into a 50 ml
container previously treated with silicone oil. 0.8 ml rat arterial
blood was injected into a vertically fixed glass tube (20 mm in
length, with an inner diameter of 4 mm and an outer diameter of 5
mm, sealed with a rubber stopper at the bottom), into which was
immediately inserted a thrombus immobilization screw made of
stainless steel. The thrombus immobilization screw, formed by
coiling of a stainless steel wire having a diameter of 0.2 mm, had
a spiral part of 18 mm in length, 15 coils each having a diameter
of 1.8 mm, and a stem of 7.0 mm in length which was connected to
the spiral part and had a question-mark-like shape. 40 min after
the blood was coagulated, the rubber stopper at the bottom of the
glass tube was removed, the stem of the thrombus immobilization
screw was nipped by forceps, and the thrombus-wrapped thrombus
immobilization screw was carefully taken out from the glass tube.
The screw was then suspended and dipped in triple-distilled water
to remove excessive blood, and accurately weighed after 1 h. The
thrombus was suspended in 8 mL of normal saline, or a solution of
compounds Ia-Il in normal saline (at a concentration of 100 nM), or
a solution of ARPAK, GRPAK, RPAK or PAK in normal saline (at a
concentration of 100 nM), or a solution of urokinase in normal
saline (100 IU/mL), then shaked at 37.degree. C. in a thermostatic
shaker (63 r/min), and removed after 2 h and accurately weighed to
determine the weight of the thrombus. The difference in thrombus
mass before and after the administration was calculated, and the
results are listed in Table 3.
[0164] As shown in the experimental results, by linking a
thrombolytic peptide ARPAK, GRPAK, RPAK or PAK and a targeting
peptide RGDS, RGDV or RGDF to a free radical scavenger TMMZ via
Lys, all compounds exhibited substantial in vitro thrombolytic
activity. Since the activity of Ia to Ic was comparable to that of
ARPAK, the activity of Id to If was comparable to that of GRPAK,
the activity of Ig to Ii was comparable to that of RPAK, and the
activity of Ij to Ii was comparable to that of PAK, on one hand the
in vitro thrombolytic activity of Ia to Il could be attributed to
the activity of the thrombolytic peptide, and on the other hand the
linking of the thrombolytic peptide ARPAK, GRPAK, RPAK or PAK and
the targeting peptide RGDS, RGDV or RGDF to the free radical
scavenger TMMZ via Lys did not abate the activity of the
thrombolytic peptide.
TABLE-US-00003 TABLE 3 In vitro thrombolytic activity by 2 h
treatment of Ia-Il Weight reduction in thrombus Compounds (Mean
.+-. SD mg) Normal saline .sup. 16.67 .+-. 1.86 .sup.a urokinase
.sup. 58.33 .+-. 4.08 .sup.a ARPAK 26.35 .+-. 3.10 Ia 28.50 .+-.
2.59 Ib 28.17 .+-. 2.31 Ic 27.33 .+-. 2.07 GRPAK 15.47 .+-. 2.61 Id
14.17 .+-. 3.55 Ie 14.00 .+-. 1.41 If 15.29 .+-. 3.36 RPAK 26.01
.+-. 3.11 Ig 27.83 .+-. 2.56 Ih 29.33 .+-. 3.01 Ii 24.83 .+-. 1.17
PAK 26.67 .+-. 3.20 Ij 26.16 .+-. 3.15 Ik 25.00 .+-. 1.54 Il 25.83
.+-. 2.31 n = 6; .sup.a p < 0.01, vs. Ia-Il
Experimental Example 4
In Vivo Thrombolysis Experiments for Compounds Ia to Il of the
Present Invention
[0165] SD rats (male, 220 to 230 g) were anaesthetized by
intraperitoneal injection of a urethane solution at a dosage of
1200 mg/kg. The anaesthetized rats were fixed in a supine position,
and the right common carotid artery was dissected, clamped at the
proximal end with an arterial clip, and penetrated with a suture at
the proximal and distal ends, respectively. The suture at the
distal end is clipped tightly by a hemostatic clamp at the fur.
Cannulation was performed at the distal end, the arterial clamp was
removed, and about 1 ml arterial blood was discharged into a 1 ml
eppendorf 0.1 ml rat arterial blood was injected into a vertically
fixed glass tube (15 mm in length, with an inner diameter of 2.5 mm
and an outer diameter of 5.0 mm, sealed with a rubber stopper at
the bottom), into which was immediately inserted a thrombus
immobilization screw made of stainless steel. The thrombus
immobilization screw, formed by coiling of a stainless steel wire
having a diameter of 0.2 mm, had a spiral part of 12 mm in length,
15 coils each having a diameter of 1.8 mm, and a stem of 1.0 mm in
length which was connected to the spiral part and had a
question-mark-like shape. 15 min after the blood was coagulated,
the rubber stopper at the bottom of the glass tube was removed, the
stem of the thrombus immobilization screw was nipped by forceps,
and the thrombus-wrapped thrombus immobilization screw was
carefully taken out of the glass tube and then accurately
weighed.
[0166] A bypass cannula was composed of 3 segments. The middle
segment was a polyethylene tubing having a length of 60.0 mm and an
inner diameter of 3.5 mm. The segments on both ends were similar
polyethylene tubes having a length of 100.0 mm, an inner diameter
of 1.0 mm and an outer diameter of 2.0 mm, one end of which was
pulled to form a tip, with an outer diameter of 1.0 mm, that could
be inserted into the rat carotid artery or vein, and the other end
of which was sheathed by a polyethylene tube having a length of 7.0
mm and an outer diameter of 3.5 mm (thickened in order to be
inserted into the polyethylene tubing of the middle segment). The
inner wall of the 3-segment cannula was entirely silylated (with 1%
silicone oil in ethyl ether). The thrombus-wrapped thrombus
immobilization screw was placed into the polyethylene tubing of the
middle segment, and both ends of the tubing sheathed the thickened
ends of the two polyethylene tubes. The cannula was filled with a
heparin solution in normal saline (50 IU/kg) through the tip end by
using an injector and was ready for use. The trachea of the
anaesthetized rat was then dissected and tracheal cannulation was
performed. The left external carotid vein of the rat was dissected,
and penetrated with a suture at the proximal and distal ends,
respectively. An uneven open incision was careful made on the
exposed left external carotid vein, and the tip of the bypass
cannula prepared as described above was inserted into the proximal
end of the open incision in the left external carotid vein, away
from the stem of the thrombus immobilization screw in the middle
segment of the bypass cannula (which accommodated the accurately
weighed thrombus immobilization screw). A precise amount of heparin
in saline (50 IU/kg) was injected through the tip at the other end
by using an injector. At this moment, without removing the injector
from the ethylene tube, the tubing between the injector and the
polyethylene tube was clamped with forceps. The blood flow was
stopped by clamping the proximal end of the right common carotid
artery with an arterial clip, and an uneven open incision was cut
carefully across the common carotid artery near the clip. The
injector was pulled out of the tip of the polyethylene tube, and
the tip of the polyethylene tube was then inserted into the
proximal end of the artery open incision. Both ends of the bypass
cannula were fixed to the artery or vein with #4 sutures.
[0167] Normal saline (3 mL/kg), or a urokinase solution in normal
saline (at a dose of 20000 IU/kg), or a solution of one of
compounds Ia-Il in normal saline (at a dose of 0.1 .mu.mol/kg), or
a solution ARPAK, GRPAK, RPAK or PAK in normal saline (at a dose of
1 .mu.mol/kg), was connected to a position close to the vein away
from the thrombus immobilization screw by using a scalp needle to
puncture the middle segment of the bypass cannula (which
accommodated the accurately weighed thrombus immobilization screw).
The artery clip was then removed to allow blood to flow from the
artery to the vein through the bypass cannula. A rat arteriovenous
bypass thrombolysis model was thus established. The solution in the
injector was slowly injected into blood, enabling normal saline
(blank control), urokinase (positive control), ARPAK, GRPAK, RPAK
or PAK (component control), or Ia-Il to act on the thrombus through
blood circulation in the order of vein-heart-artery. The process
was timed at the beginning of injection, and the thrombus
immobilization screw was removed from the bypass cannula after 1 h
and accurately weighed. The difference in the mass of the thrombus
immobilization screw in the rat bypass cannula before and after the
administration was determined, and the experimental results are
shown in Table 4.
[0168] As shown in the experimental results, not only did compounds
Ia-Ic, obtained by linking a thrombolytic peptide ARPAK, GRPAK,
RPAK or PAK and a targeting peptide RGDS, RGDV or RGDF to a free
radical scavenger TMMZ via Lys, exhibit thrombolytic activity at a
dosage of 0.1 .mu.mol/kg, the potency of their activity was also
comparable to that of the corresponding thrombolytic peptide ARPAK,
GRPAK, RPAK or PAK at a dosage of 1 .mu.mol/kg. As such, by linking
the thrombolytic peptide ARPAK, GRPAK, RPAK or PAK and the
targeting peptide RGDS, RGDV or RGDF to the free radical scavenger
TMMZ via Lys, the effective dosage could be decreased by 10
folds.
TABLE-US-00004 TABLE 4 In vivo thrombolytic activity of Ia-Il
Weight reduction in thrombus Compounds (Mean .+-. SD mg) Normal
saline .sup. 11.05 .+-. 1.51 .sup.a urokinase .sup. 18.02 .+-. 2.32
.sup.a ARPAK 15.20 .+-. 2.55 Ia 15.39 .+-. 3.19 Ib 14.35 .+-. 2.95
Ic 15.79 .+-. 3.07 GRPAK 15.47 .+-. 2.61 Id 14.17 .+-. 3.55 Ie
14.00 .+-. 1.41 If 15.29 .+-. 3.36 RPAK 15.67 .+-. 2.61 Ig 16.35
.+-. 2.42 Ih 15.37 .+-. 1.82 Ii 15.73 .+-. 2.95 PAK 15.00 .+-. 2.61
Ij 14.89 .+-. 1.84 Ik 15.47 .+-. 2.61 Il 16.21 .+-. 2.84 n = 10;
.sup.a p < 0.01 vs. Ia-Il
Experimental Example 5
In Vivo Anti-Thrombus Experiments for Compounds Ia to Il of the
Present Invention
[0169] SD rats (male, 220 to 230 g) were randomly divided into
groups with 11 rats in each group. The rats were fed at a resting
state for 1 day and fasted overnight. The rats were given normal
saline (at a dose of 3 mL/kg), a solution of one of compounds Ia-Il
in normal saline (at a dose of 0.1 mmol/kg), a solution of the
targeting peptide RGDS, RGDV or RGDF in normal saline (at a dose of
10 .mu.mol/kg), or aspirin (at a dose of 33 mg/kg) by gavage. After
30 min, the rats were anesthetized with a 20% urethane solution,
and the right carotid artery and the left carotid vein were
dissected. A cannula was filled with sodium heparin in normal
saline, and one end thereof was inserted into the left vein, while
the other end was injected with a certain amount of sodium heparin
for anticoagulation with an injector and then inserted into the
right artery. Blood flew from the right artery to the left vein
through the polyethylene tubing, and the thread attached with
thrombus was taken out after 15 min and the weight thereof was
recorded. The wet thrombus weight was determined by subtracting the
thread weight from the total weight. The results are shown in Table
5.
[0170] As shown in the experimental results, not only did compounds
Ia-Ic, obtained by linking a thrombolytic peptide ARPAK, GRPAK,
RPAK or PAK and a targeting peptide RGDS, RGDV or RGDF to a free
radical scavenger TMMZ via Lys, exhibit anti-thrombus activity at
an oral dosage of 0.1 .mu.mol/kg, the potency of their activity was
also comparable to that of the targeting peptide RGDS, RGDV or RGDF
at a dosage of 10 .mu.mol/kg. As such, by linking the thrombolytic
peptide ARPAK, GRPAK, RPAK or PAK and the targeting peptide RGDS,
RGDV or RGDF to the free radical scavenger TMMZ via Lys, the
effective dosage could be decreased by 100 folds.
TABLE-US-00005 TABLE 5 In vivo anti-thrombus activity of Ia-Il Wet
weight of thrombus Compounds (Mean .+-. SD mg) normal saline .sup.
27.38 .+-. 2.62 .sup.a aspirin .sup. 12.85 .+-. 2.49 .sup.a RGDS
20.02 .+-. 2.35 RGDV 21.26 .+-. 2.07 RGDF 19.55 .+-. 2.21 Ia 24.32
.+-. 2.10 Ib 20.14 .+-. 2.45 Ic 20.50 .+-. 2.26 Id 19.46 .+-. 1.84
Ie 16.92 .+-. 1.53 If 17.99 .+-. 2.47 Ig 17.89 .+-. 2.05 Ih 18.24
.+-. 1.89 Ii 17.79 .+-. 2.02 Ij 19.45 .+-. 1.79 Ik 22.25 .+-. 2.25
Il 19.32 .+-. 2.56 n = 11; .sup.a p < 0.01 vs. Ia-Il
Establishment of Animal Models for Assessment of the Efficacy of
the Compounds According to the Present Invention in Treating Stroke
Patients
[0171] (1) The rat experimental protocol described herein was in
accordance with the Geneva guidance in animal experiments and
approved by the college ethical committee. Clean grade health male
SD rats, weighing 280 to 305 g, were purchased from Vital River
Laboratories of Experimental Animals. These rats were randomly used
for preparation of thrombus or establishment of stroke models.
[0172] (2) A 10% chloral hydrate solution was injected
intraperitoneally into SD rats at a dosage of 400 mg/kg body weight
for anesthesia. The carotid artery was dissected, 15 mL fresh
arterial blood was drawn, and aliquots of 10 .mu.L each were then
added into 1.5 mL EP vials. The thrombus formed was kept at RT for
2 h and then in a -20.degree. C. refrigerator for 22 h. When used,
0.5 mL saline was added to the thrombus which was broken up by
using a glass rod, so as to prepare a thrombus homogenate
suspension solution, with a volume of about 0.1 mm.sup.3 for each
thrombus pieces.
[0173] (3) A 10% chloral hydrate solution was injected
intraperitoneally into SD rats at a dosage of 400 mg/kg body weight
for anesthesia. The carotid artery was dissected, 15 mL fresh
arterial blood was drawn, and aliquots of 10 .mu.L each were then
added into 1.5 mL EP vials. The thrombus formed was firstly kept at
RT for 24 h. When used, 0.5 mL saline was added to the thrombus
which was broken up by using a glass rod, so as to prepare a
thrombus homogenate suspension solution, with a volume of about 0.1
mm.sup.3 for each thrombus pieces.
[0174] (4) A 10% chloral hydrate solution was injected
intraperitoneally into male SD rats at a dosage of 400 mg/kg body
weight for anesthesia. A longitudinal open incision was made at the
center of the neck, and the right common carotid artery trunk was
dissected (about 3 cm in length). Carotid external artery branches
were each dissected and ligated at the hyoid level, and the carotid
internal artery was dissected at the swollen part of the neck. The
open incisions in the carotid internal artery and the proximal end
of the common carotid artery were occluded respectively with
noninvasive arterial clips, and the distal end of the carotid
external artery was ligated. A catheter containing 0.5 mL thrombus
suspension in normal saline was inserted in the carotid external
artery trunk. At the same time when the clip on the carotid
internal artery was released, the 0.5 mL thrombus suspension in
normal saline in the catheter slowly flew from the carotid external
artery to its proximal end, and then was injected into the arteries
in brain through the carotid internal artery. Subsequently, the
proximal end of the carotid internal artery was ligated, the
arterial clips on the carotid internal artery and the common
carotid artery were released, and blood flow was restored. The main
jugular external vein was dissected, and normal saline (blank
control) or a solution of the compounds of the present invention in
normal saline was infused through the jugular external vein. After
the wound was stitched up, 20,000 IU penicillin was intramuscularly
injected for prevention from infection. The model of immediate
treatment after the onset of stroke was thus established.
[0175] (5) A 10% chloral hydrate solution was injected
intraperitoneally into male SD rats at a dosage of 400 mg/kg body
weight for anesthesia. A longitudinal open incision was made at the
center of the neck, and the right common carotid artery trunk was
dissected (about 3 cm in length). Carotid external artery branches
were each dissected and ligated at the hyoid level, and the carotid
internal artery was dissected at the swollen part of the neck. The
open incision in the carotid internal artery and the proximal end
of the common carotid artery were occluded respectively with
noninvasive arterial clips, and the distal end of the carotid
external artery was ligated. A catheter containing 0.5 mL thrombus
suspension in normal saline was inserted in the carotid external
artery trunk. At the same time when the clip on the carotid
internal artery was released, the 0.5 mL thrombus suspension in
normal saline in the catheter slowly flew from the carotid external
artery to its proximal end, and then was injected into the arteries
in brain through the carotid internal artery. Subsequently, the
proximal end of the carotid artery was ligated, the arterial clips
on the carotid internal artery and the common carotid artery were
released, and blood flow was restored. After the wound was stitched
up, 20,000 IU penicillin was intramuscularly injected for
prevention from infection. After 4 h, 6 h or 24 h, normal saline
(blank control) or a solution of the compounds of the present
invention in normal saline was infused through the tail vein. The
rat models of 4 h, 6 h, and 24 h post-onset treatment of stroke
were thus established.
Establishment of Animal Models for Assessment of the Efficacy of
the Compounds According to the Present Invention after 4 h, 6 h,
and 24 h from Stroke Onset
[0176] (1) The efficacy after immediate, 4 h post-onset treatment,
and 6 h post-onset treatment of stroke rats with the compounds
according to the present invention means the result of scoring of
rats' behaviors 24 h after the rats regained consciousness. The
behaviors include the walking manner, the degree of drooping of the
right eye lid, the degree of tail stiffness, tension of muscles,
the degree of head tilting, the support force of limbs, and the
death status.
[0177] (2) The efficacy in 24 h post-onset treatment of stroke in
rats with the compounds according to the present invention means
the result of observation of rats' behaviors 24 h after the rats
regained consciousness. The behaviors include the walking manner,
the degree of drooping of the right eye lid, the degree of tail
stiffness, tension of muscles, the degree of head tilting, the
support force of limbs, and the death status.
[0178] (3) The efficacy in rats with stroke treated once with the
compound of the present invention was compared to the efficacy in
rats with stroke treated once with saline.
[0179] (4) Rats with stroke with continuous treatment were injected
with the compounds of the present invention in normal saline every
24 h through the tail vein. On the next day, the videos were
recorded, and comparison was made among the recorded results.
Test Results of the Compounds Ia to Il According to the Present
Invention in the Above Animal Models are as Follows:
Experimental Example 6
Experiments on Rats that Received Immediate Treatment after Stroke
Onset with Compounds Ia to Il of the Present Invention
[0180] The in vivo anti-stroke activity of the present invention
was represented by neural function scores, with a lower score
indicating higher activity. A 10% chloral hydrate solution (400
mg/kg) was injected intraperitoneally into SD male rats (250-300 g)
for anesthesia. An open incision of 2 cm in length was
longitudinally made slightly on the right to the center of the
neck, and the right common carotid artery trunk, carotid external
artery and carotid internal artery were dissected along the margin
of the inner side of sternocleidomastoid muscles. The open
incisions in the carotid internal artery and the proximal end of
the common carotid artery were occluded respectively with
noninvasive arterial clips. A small open incision was made across
the carotid external artery, and the distal end of the carotid
external artery was ligated. The arterial clip at the proximal end
of the carotid external artery was released, and 10 .mu.l blood was
drawn before the proximal end of the common carotid artery was
again occluded with the noninvasive arterial clip. The 10 .mu.l
blood drawn was placed in a 1 mL EP vial and kept at RT for 30 min
for coagulation of blood, and then transferred into a -20.degree.
C. refrigerator for 1 h to allow coagulation. After 1 h, the blood
clots were taken out, into which 1 mL saline was added, and then
broken into uniform microthrombus by using a steel spatula. The
microthrombus suspension was then transferred to a 1 mL injector
until use. At the same time when the clip on the carotid internal
artery of the rat was released, 1 mL thrombus suspension in the
injector was slowly injected from the carotid external artery of
the rat to its proximal end, and the suspension was injected into
the brain of the rat through the carotid internal artery.
Subsequently, the proximal end of the carotid external artery was
ligated, the arterial clips on the carotid internal artery and the
common carotid artery were released, and blood flow was restored.
The jugular common vein of the rats was dissected. The vein was
immediately ligated, 3 drops of penicillin was dropped at the wound
site, the wound was stitched up, and the animals were allowed to
come around, as the sham operation group. Or injection of urokinase
in normal saline (positive control group, at a dosage of 20000
IU/kg), normal saline (blank control group, at a dosage of 3
ml/kg), TMMZ in normal saline (component control group, at a dosage
of 1 .mu.mol/kg), a thrombolytic peptide ARPAK, GRPAK, RPAK or PAK
in normal saline (component control group, at a dosage of 1
.mu.mol/kg), or one of compounds Ia-Il in normal saline (at a
dosage of 0.1 .mu.mol/kg) was carried out. 24 h after the rats were
awake, the degree of damage in the neural function was evaluated by
the Zealonga method. A score of 0 indicated no sign of loss in
neural function, 1 indicated the front limbs on the undamaged side
could not stretch out, 2 indicated walking toward the undamaged
side, 3 indicated tail-chasing walking in circles toward the
undamaged side, 4 indicated involuntary walking with disturbance of
consciousness, and 5 indicated death. The experimental results are
shown in Table 6.
[0181] As shown in the experimental results, the compounds Ia-Ic,
obtained by linking a thrombolytic peptide ARPAK, GRPAK, RPAK or
PAK and a targeting peptide RGDS, RGDV or RGDF to a free radical
scavenger TMMZ via Lys, exhibited anti-stroke activity at a dosage
of 0.1 .mu.mol/kg, whereas urokinase did not exhibit anti-stroke
activity at a dosage of 20000 IU/kg. Similarly, the thrombolytic
peptide ARPAK, GRPAK, RPAK or PAK did not exhibit anti-stroke
activity at a dosage of 1 .mu.mol/kg. As such, by linking the
thrombolytic peptide ARPAK, GRPAK, RPAK or PAK and the targeting
peptide RGDS, RGDV or RGDF to the free radical scavenger TMMZ via
Lys, the compounds were provided with an anti-stroke function.
Specially, at the dosage of 1 mmol/kg, 4 compounds had anti-stroke
activity comparable to that of urokinase at a dosage of 20000
IU/kg, and 8 compounds had remarkably higher anti-stroke activity
than that of urokinase at a dosage of 20000 IU/kg.
TABLE-US-00006 TABLE 6 In vivo anti-stroke activity of Ia-Ic Neural
function scores Compounds (Mean .+-. SD) normal saline 3.07 .+-.
1.04 .sup. urokinase 1.90 .+-. 1.37 .sup.a TMMZ 2.83 .+-. 0.75
.sup.a ARPAK 2.21 .+-. 0.94 .sup.a Ia 1.00 .+-. 1.01 .sup.b Ib 0.56
.+-. 1.01 .sup.c Ic 0.89 .+-. 1.36 .sup.c GRPAK 2.38 .+-. 0.92
.sup.a Id 1.22 .+-. 1.32 .sup.b Ie 0.44 .+-. 1.01 .sup.c If 0.60
.+-. 0.84 .sup.c RPAK 2.38 .+-. 0.97 .sup.a Ig 1.00 .+-. 1.19
.sup.b Ih 1.33 .+-. 1.22 .sup.b Ii 0.87 .+-. 1.05 .sup.c PAK 2.42
.+-. 0.95 .sup.a Ij 0.90 .+-. 1.10 .sup.c Ik 0.56 .+-. 0.53 .sup.c
Il 0.50 .+-. 0.53 .sup.c n = 10; .sup.a p > 0.05 vs. normal
saline; .sup.b p > 0.05 vs. urokinase; p < 0.01 vs. normal
saline; .sup.c p < 0.01 vs. normal saline or urokinase
Experimental Example 7
Experiments on Cerebral Infarction Volume in Rats that Received
Immediate Treatment with Compounds Ia to Il of the Present
Invention after the Stroke Onset
[0182] After the rats were awake for 24 h and assessed for their
degree of damage in neural function in Experimental example 6, they
were anesthesized with urethane followed by immediate decapitation
and removal of the brain. Brain tissues were kept in a -20.degree.
C. refrigerator for 2 h, and coronal sections of about 2 mm were
successively sliced from the prefrontal end for a total of 6
sections, and then placed into a 2% TTC solution to incubate in
darkness at 37.degree. C. for 30 min. The color change in brain
sections was observed: normal brain tissues were stained red by
TTC, while ischemic brain tissues, i.e., brain tissues with
infracts, appeared in a white color. Photographs were taken by
using a digital camera and processed with SPSS statistics software,
and the volume of infarction in brain tissues and the volume of
normal brain tissues in the coronal sections were calculated. The
experimental results are shown in Table 7.
[0183] As shown in the experimental results, not only did compounds
Ia-Ic, obtained by linking a thrombolytic peptide ARPAK, GRPAK,
RPAK or PAK and a targeting peptide RGDS, RGDV or RGDF to a free
radical scavenger TMMZ via Lys, exhibit an effect in reduction of
the cerebral infarction volume in rats with stroke at a dosage of
0.1 .mu.mol/kg, such an effect was substantially more potent than
that of urokinase at a dosage of 20000 IU/kg.
TABLE-US-00007 TABLE 7 Volume of cerebral infarction in stroke rats
treated with Ia-Ic Percentage of infarction volume Compounds (Mean
.+-. SD %) normal saline 22.92 .+-. 2.74 urokinase .sup. 11.00 .+-.
2.42 .sup.b TMMZ .sup. 22.96 .+-. 2.43 .sup.a ARPAK .sup. 22.00
.+-. 2.20 .sup.a Ia 7.21 .+-. 0.82 Ib 7.13 .+-. 0.83 Ic 7.40 .+-.
1.65 GRPAK .sup. 21.77 .+-. 2.46 .sup.a Id 8.21 .+-. 1.91 Ie 6.44
.+-. 1.51 If 7.47 .+-. 1.31 RPAK .sup. 22.11 .+-. 2.25 .sup.a Ig
6.40 .+-. 0.28 Ih 7.35 .+-. 1.14 Ii 7.06 .+-. 1.08 PAK .sup. 22.07
.+-. 2.40 .sup.a Ij 6.84 .+-. 0.82 Ik 7.86 .+-. 1.02 Il 6.56 .+-.
0.41 n = 10; .sup.a p > 0.05, vs. normal saline; .sup.b p <
0.01 vs. normal saline and Ia-Ic
Experimental Example 8
Experiments on Rats that Received Immediate Treatment with
Different Dosages of Compound Ie of the Present Invention after the
Stroke Onset
[0184] After analysis and comparison of all experimental results in
the present invention, compound Ie was used as the representative,
in order to demonstrate the dose-dependent therapeutic effect
exhibited by compounds Ia to Il in the above experiments. It should
be noted that other compounds of Ia to Il could achieve similar
dose-dependent therapeutic effect as compound Ie did, since the
other compounds of Ia to Il had achieved the same effect as
compound Ie in NO free radical scavenging, euglobulin clot lysis,
thrombolysis, anti-thrombus action, and treatment of stroke in
rats.
[0185] A 10% chloral hydrate solution (400 mg/kg) was injected
intraperitoneally into male SD rats (250 to 300 g) for anesthesia.
An incision of about 2 cm in length was longitudinally made
slightly on the right to the center of the neck, and the right
carotid common artery, carotid external artery and carotid internal
artery were dissected along the margin of the inner side of
sternocleidomastoid muscles. The open incision in the carotid
internal artery and the proximal end of the common carotid artery
were occluded respectively with noninvasive arterial clips. A small
incision was made on the carotid external artery, and the distal
end of the carotid external artery was ligated. The arterial clip
at the proximal end of the carotid external artery was released,
and 10 .mu.l blood was drawn before the proximal end of the common
carotid artery was again occluded with a noninvasive arterial clip.
The 10 .mu.l blood drawn was placed in a 1 mL EP vial and kept at
RT for 30 min until coagulation of blood, and then transferred into
a -20.degree. C. refrigerator for 1 h to allow solid coagulation.
After 1 h, the blood clots were taken out, added into 1 mL saline,
and then broken into relatively uniform microthrombus by using a
steel spatula. The microthrombus suspension was then transferred
into a 1 mL injector until use. At the same time when the clip on
the carotid internal artery of the rat was released, the 1 mL
thrombus suspension in the injector was slowly injected from the
carotid external artery of the rat to its proximal end, and then
was injected into the brain of the rat through the carotid internal
artery. Subsequently, the proximal end of the carotid external
artery was ligated, the arterial clips on the carotid internal
artery and the carotid common artery were released, and blood flow
was restored.
[0186] Injection of urokinase in normal saline (positive control
group, at a dosage of 20000 IU/kg), tPA in normal saline (positive
control group, at a dosage of 3 mg/kg), normal saline (blank
control group, at a dosage of 3 mL/kg), or compound Ie in normal
saline (at a dosage of 1 .mu.mol/kg, 0.1 .mu.mol/kg or 0.1
.mu.mol/kg) was carried out. 24 h after the rats were awake, the
degree of damage in neural function was evaluated by the Zealonga
method. A score of 0 indicated no sign of loss in neural function,
1 indicated the front limbs on the undamaged side could not stretch
out, 2 indicated walking toward the undamaged side, 3 indicated
tail-chasing walking in circles toward the undamaged side, 4
indicated involuntary walking with disturbance of consciousness,
and 5 indicated death. The experimental results are shown in Table
8. As shown in the results, in rats receiving immediate treatment
after the onset of stroke with 1 .mu.mol/kg, 0.1 .mu.mol/kg and
0.01 .mu.mol/kg compound Ie, the percentage of rats with a neural
function score of 0 was 60%, 30%, and 0%, respectively; and the
percentage of rats with a neural function score of 1 was 20%, 30%,
and 10%, respectively. Thus, it shows that the anti-stroke activity
of compound Ie was dose-dependent. Further, in rats with stroke
treated with 20000 IU/kg urokinase and 3 mg/kg tPA, the percentage
of rats with a neural function score of 0 was 10% and 40%,
respectively, and the percentage of rats with a neural function
score of 1 was 50% and 10%, respectively; in comparison, the
efficacy of 1 .mu.mol/kg and 0.1 .mu.mol/kg compound Ie was
obviously superior.
TABLE-US-00008 TABLE 8 In vivo anti-stroke activity of compound Ie
of the present invention at different dosages Daily neural function
scores (Mean .+-. SD) and number of rats scored Compounds Score 0
Score 1 Score 2 Score 3 Score 4 Score 5 normal saline 0 2 3 5 1 0
urokinase 1 5 0 3 1 0 tPA 4 1 1 3 1 0 Ie 1 .mu.nmol/kg 6 2 0 2 0 0
100 nmol/kg 3 3 0 3 1 0 10 nmol/kg 0 1 6 1 1 0 n = 10; a) p <
0.01 vs. normal saline
Experimental Example 9
Experiments on Rats Receiving 6 Successive Treatments with 1
.mu.mol/kg Compound Ie of the Present Invention 4 Hours after the
Onset of Stroke
[0187] The efficacy was represented by neural function scores, and
a lower score indicates higher efficacy. A 10% chloral hydrate
solution was injected intraperitoneally into male SD rats at a
dosage of 400 mg/kg body weight for anesthesia. A longitudinal
incision was made at the center of the neck, and the right carotid
common artery trunk was dissected (about 3 cm in length). Carotid
external artery branches were each dissected and ligated at the
hyoid level, and the carotid internal artery was dissected at the
swollen part of the neck. The open incision in the carotid internal
artery and the proximal end of the common carotid artery were
occluded respectively with noninvasive arterial clips, and the
distal end of the carotid external artery was ligated. A catheter
containing 0.5 mL thrombus suspension in normal saline was inserted
in the carotid external artery trunk. At the same time when the
clip on the carotid internal artery was released, the 0.5 mL
thrombus suspension in normal saline in the catheter slowly flew
from the carotid external artery to its proximal end, and then was
injected into the arteries in brain through the carotid internal
artery. Subsequently, the proximal end of the carotid artery was
ligated, the arterial clips on the carotid internal artery and the
common carotid artery were released, and blood flow was restored.
After the wound was stitched up, 20,000 IU penicillin was
intramuscularly injected for prevention from infection. After 4 h,
compound Ie in normal saline (at a dosage of 1 .mu.mol/kg, n=11),
urokinase in normal saline (at a dosage of 20000 IU/kg, n=6) or tPA
in normal saline (at a dosage of 3 mg/kg, n=6) was infused through
the tail vein. Infusion of compound Ie in normal saline through rat
tail vein was carried out once per day for 6 consecutive days,
observed for 7 days. The rats were compared to themselves each day,
and evaluated for degree of damage in neural function by the
Zealonga method. Alternatively, infusion of urokinase in normal
saline through rat tail vein was carried out once per day for two
consecutive days, the rats were compared to themselves each day,
and evaluated for the degree of damage in neural function by the
Zealonga method. Alternatively, infusion of tPA in normal saline
through rat tail vein was carried out once per day for two
consecutive days, the rats were compared to themselves each day,
and evaluated for the degree of damage in neural function by the
Zealonga method. A score of 0 indicated no sign of loss in neural
function, 1 indicated the front limbs on the undamaged side could
not stretch out, 2 indicated walking toward the undamaged side, 3
indicated tail-chasing walking in circles toward the undamaged
side, 4 indicated involuntary walking with disturbance of
consciousness, and 5 indicated death. The experimental results are
shown in Tables 9-1, 9-2 and 9-3.
[0188] The data in Table 9-1 demonstrated that, in rats that
received treatment 4 h after the onset of stroke with one dose of 1
.mu.mol/kg compound Ie each day for 6 consecutive days, excluding
one that accidentally died on day 2, 8 out of the remaining 10 rats
recovered to have no sign of loss in neural function while the rest
2 rats had only the sign of slight loss in neural function. Thus,
compound Ie exhibited therapeutic effect at a dosage of 1
.mu.mol/kg in stroke beyond the golden treatment window.
TABLE-US-00009 TABLE 9-1 Efficacy in rats receiving treatment with
1 .mu.mol/kg compound Ie of the present invention 4 h after the
onset of stroke Daily neural function scores Time of (Mean .+-. SD)
and number of rats scored scoring Score 0 Score 1 Score 2 Score 3
Score 4 Score 5 Day 1 1 rat 4 rats 4 rats 1 rat 1 rat 0 Day 2 3
rats 5 rats 1 rat 1 rat 0 1 rat Day 3 5 rats 5 rats 0 0 0 0 Day 4 7
rats 3 rats 0 0 0 0 Day 5 8 rats 2 rats 0 0 0 0 Day 6 8 rats 2 rats
0 0 0 0 Day 7 8 rats 2 rats 0 0 0 0
[0189] The data in Table 9-2 demonstrated that, in rats that
received treatment 4 h after the onset of stroke with one dose of
20000 IU/kg urokinase each day, 2 out of 6 rats died within 48 h.
Upon autopsy on the dead rats, both showed hemorrhage in internal
organs, particularly severe hemorrhage in lungs. Therefore, the
dosage regime was discontinued after two doses. No rats after
receiving two doses recovered to have no sign of loss in neural
function or to have only the sign of slight loss in neural
function.
TABLE-US-00010 TABLE 9-2 Efficacy in rats receiving treatment with
20000 IU/kg urokinase 4 h after the onset of stroke Daily neural
function scores Time of (Mean .+-. SD) and number of rats scored
scoring Score 0 Score 1 Score 2 Score 3 Score 4 Score 5 Day 1 2
rats 3 rats 1 rat Day 2 3 rats 1 rat 1 rat
[0190] The data in Table 9-3 demonstrated that, in rats that
received treatment 4 h after the onset of stroke with one dose of 3
mg/kg tPA each day, 1 out of 6 rats died within 24 h. Upon autopsy
on the dead rat, it showed hemorrhage in internal organs,
particularly severe hemorrhage in lungs. Therefore, the dosage
regime was discontinued after two doses. No rats after receiving
two doses recovered to have no sign of loss in neural function, and
2 rats recovered to have only the sign of slight loss in neural
function.
TABLE-US-00011 TABLE 9-3 Efficacy in rats receiving treatment with
3 mg/kg tPA 4 h after the onset of stroke Daily neural function
scores Time of (Mean .+-. SD) and number of rats scored scoring
Score 0 Score 1 Score 2 Score 3 Score 4 Score 5 Day 1 2 rats 3 rats
1 rat Day 2 2 rats 2 rats 1 rat
[0191] In summary of the data in Table 9-1, 9-2 and 9-3, even for
rats that received treatment 4 h after the onset of stroke for two
consecutive days, compound Ie at a dosage of 1 .mu.mol/kg showed
much higher efficacy than urokinase at a dosage of 20000 IU/kg and
tPA at a dosage of 3 mg/kg.
Experimental example 10
Experiments on Rats Receiving 6 Successive Treatments with 1
.mu.mol/kg Compound Ie of the Present Invention 6 h after the Onset
of Stroke
[0192] The efficacy was represented by neural function scores, and
a lower score indicates higher efficacy. A 10% chloral hydrate
solution was injected intraperitoneally into male SD rats at a
dosage of 400 mg/kg body weight for anesthesia. A longitudinal open
incision was made at the center of the neck, and the right common
carotid artery trunk was dissected (about 3 cm in length). Carotid
external artery branches were each dissected and ligated at the
hyoid level, and the carotid internal artery was dissected at the
swollen part of the neck. The open incision in the carotid internal
artery and the proximal end of the common carotid artery were
occluded respectively with noninvasive arterial clips, and the
distal end of the carotid external artery was ligated. A catheter
containing 0.5 mL thrombus suspension in normal saline was inserted
in the carotid external artery trunk. At the same time when the
clip on the carotid internal artery was released, the 0.5 mL
thrombus suspension in normal saline in the catheter slowly flew
from the carotid external artery to its proximal end, and then was
injected into the arteries in brain through the carotid internal
artery. Subsequently, the proximal end of the carotid artery was
ligated, the arterial clips on the carotid internal artery and the
common carotid artery were released, and blood flow was restored.
After the wound was stitched up, 20,000 IU penicillin was
intramuscularly injected for prevention from infection. After 6 h,
compound Ie in normal saline (at a dosage of 1 .mu.mol/kg, n=11),
urokinase in normal saline (at a dosage of 20000 IU/kg, n=6) or tPA
in normal saline (at a dosage of 3 mg/kg, n=6) was infused through
the tail vein. Infusion of compound Ie in normal saline through rat
tail vein was carried out once per day for 6 consecutive days,
observed for 7 days. The rats were compared to themselves each day,
and evaluated for the degree of damage in neural function by the
Zealonga method. Alternatively, infusion of urokinase in normal
saline through rat tail vein was carried out once per day for two
consecutive days, the rats were compared to themselves each day,
and evaluated for the degree of damage in neural function by the
Zealonga method. Alternatively, infusion of tPA in normal saline
through rat tail vein was carried out once per day for two
consecutive days, the rats were compared to themselves each day,
and evaluated for the degree of damage in neural function by the
Zealonga method. A score of 0 indicated no sign of loss in neural
function, 1 indicated the front limbs on the undamaged side could
not stretch out, 2 indicated walking toward the undamaged side, 3
indicated tail-chasing walking in circles toward the undamaged
side, 4 indicated involuntary walking with disturbance of
consciousness, and 5 indicated death. The experimental results are
shown in Tables 10-1, 10-2 and 10-3.
[0193] The data in Table 10-1 demonstrated that, in rats that
received treatment 6 h after the onset of stroke with one dose of 1
.mu.mol/kg compound Ie each day for 6 consecutive days, excluding
two that accidentally died on day 2, 2 out of the remaining 9 rats
recovered to have no sign of loss in neural function, one rat
recovered to have only the sign of slight loss in neural function,
and 6 showed the sign of tail-chasing walking in circles toward the
undamaged side. Thus, compound Ie exhibited therapeutic effect at a
dosage of 1 .mu.mol/kg in stroke beyond the golden treatment
window.
TABLE-US-00012 TABLE 10-1 Efficacy in rats receiving treatment with
1 .mu.mol/kg compound Ie of the present invention 6 h after the
onset of stroke Daily neural function scores Time of (Mean .+-. SD)
and number of rats scored scoring Score 0 Score 1 Score 2 Score 3
Score 4 Score 5 Day 1 0 3 rats 5 rats 2 rats 1 rat 0 Day 2 0 2 rats
2 rats 4 rats 1 rat 2 rats Day 3 0 2 rats 4 rats 2 rats 1 rat 0 Day
4 1 rat 1 rat 2 rats 4 rats 0 0 Day 5 1 rat 1 rat 2 rats 4 rats 0 0
Day 6 2 rats 1 rat 1 rat 4 rats 1 rat 0 Day 7 2 rats 1 rat 0 6 rats
0 0
[0194] The data in Table 10-2 demonstrated that, in rats that
received treatment 6 h after the onset of stroke with one dose of
20000 IU/kg urokinase each day, 4 out of 6 rats died within 24 h.
Upon autopsy on the dead rats, all showed hemorrhage in internal
organs, particularly severe hemorrhage in lungs. Therefore, the
dosage regime was discontinued after two doses. One rat after
receiving two doses recovered to have no sign of loss in neural
function, and one rat showed the sign of involuntary walking with
disturbance of consciousness.
TABLE-US-00013 TABLE 10-2 Efficacy in rats receiving treatment with
20000 IU/kg urokinase 6 h after the onset of stroke Daily neural
function scores Time of (Mean .+-. SD) and number of rats scored
scoring Score 0 Score 1 Score 2 Score 3 Score 4 Score 5 Day 1 1 rat
1 rat 4 rats Day 2 1 rat 1 rat
[0195] The data in Table 10-3 demonstrated that, in rats that
received treatment 6 h after the onset of stroke with one dose of 3
mg/kg tPA each day, 2 out of 6 rats died within 24 h. Upon autopsy
on the dead rats, both showed hemorrhage in internal organs,
particularly severe hemorrhage in lungs. Therefore, the dosage
regime was discontinued after two doses. No rats after receiving
two doses recovered to have no sign of loss in neural function, 2
rats recovered to have only the sign of slight loss in neural
function, one rat showed the sign of tail-chasing walking in
circles toward the undamaged side, and one rat showed the sign of
involuntary walking with disturbance of consciousness.
TABLE-US-00014 TABLE 10-3 Efficacy in rats receiving treatment with
3 mg/kg tPA 6 h after the onset of stroke Daily neural function
scores Time of (Mean .+-. SD) and number of rats scored scoring
Score 0 Score 1 Score 2 Score 3 Score 4 Score 5 Day 1 1 rat 1 rat 1
rat 1 rat 2 rats Day 2 2 rats 1 rat 1 rat
[0196] In summary of the data in Table 10-1, 10-2 and 10-3, even
for rats that received treatment 6 h after the onset of stroke for
two consecutive days, compound Ie at a dosage of 1 .mu.mol/kg
showed much higher efficacy than urokinase at a dosage of 20000
IU/kg and tPA at a dosage of 3 mg/kg.
Experimental Example 11
Experiments on Rats Receiving Treatments 6 h after the Onset of
Stroke with Compound Ie of the Present Invention at an Initial
Dosage of 5 .mu.mol/kg and 5 Subsequent Dosages of 2 .mu.mol/kg
Each
[0197] The efficacy was represented by neural function scores, and
a lower score indicates higher efficacy. A 10% chloral hydrate
solution was injected intraperitoneally into male SD rats at a
dosage of 400 mg/kg body weight for anesthesia. A longitudinal open
incision was made at the center of the neck, and the right common
carotid artery trunk was dissected (about 3 cm in length). Carotid
external artery branches were each dissected and ligated at the
hyoid level, and the carotid internal artery was dissected at the
swollen part of the neck. The open incision in the carotid internal
artery and the proximal end of the common carotid artery were
occluded respectively with noninvasive arterial clips, and the
distal end of the carotid external artery was ligated. A catheter
containing 0.5 mL thrombus suspension in normal saline was inserted
in the carotid external artery trunk. At the same time when the
clip on the carotid internal artery was released, the 0.5 mL
thrombus suspension in normal saline in the catheter slowly flew
from the carotid external artery to its proximal end, and then was
injected into the arteries in brain through the carotid internal
artery. Subsequently, the proximal end of the carotid artery was
ligated, the arterial clips on the carotid internal artery and the
common carotid artery were released, and blood flow was restored.
After the wound was stitched up, 20,000 IU penicillin was
intramuscularly injected for prevention from infection. After 6 h,
compound Ie in normal saline (at an initial dosage of 5 .mu.mol/kg,
n=12) was infused through the tail vein. Then, infusion of compound
Ie in normal saline (at a dosage of 2 .mu.mol/kg, n=12) through rat
tail vein was carried out once per day for 6 consecutive days,
observed for 7 days. The rats were compared to themselves each day,
and evaluated for the degree of damage in neural function by the
Zealonga method. A score of 0 indicated no sign of loss in neural
function, 1 indicated the front limbs on the undamaged side could
not stretch out, 2 indicated walking toward the undamaged side, 3
indicated tail-chasing walking in circles toward the undamaged
side, 4 indicated involuntary walking with disturbance of
consciousness, and 5 indicated death. The experimental results are
shown in Table 11.
[0198] The data in Table 11 demonstrated that, efficacy was shown
in rats that received treatment 6 h after the onset of stroke with
one dose of 5 .mu.mol/kg compound Ie on day 1 and one dose of 2
mmol/kg compound Ie per day for the following 5 days. Among the 12
rats that received the treatment, two were dead, while 6 out of the
remaining 10 rats recovered to have no sign of loss in neural
function, two had only the sign of slight loss in neural function,
one showed the sign of walking toward the undamaged side, and one
showed the sign of tail-chasing walking in circles toward the
undamaged side. Thus, continuous treatment with compound Ie showed
therapeutic effect on stroke beyond the golden treatment
window.
TABLE-US-00015 TABLE 11 Efficacy in rats receiving treatment with
compound Ie of the present invention 6 h after the onset of stroke
Daily neural function scores (Mean .+-. SD) Rat No. Day 1 Day 2 Day
3 Day 4 Day 5 Day 6 Day 7 1 1 1 0 0 0 0 0 2 1 0 0 0 0 0 0 3 3 1 5 4
2 1 1 0 0 0 0 5 0 0 0 0 0 0 0 6 2 2 1 0 0 0 0 7 5 8 2 1 1 1 1 1 1 9
0 0 0 0 0 0 0 10 4 3 3 2 2 2 2 11 3 3 3 2 1 1 1 12 1 1 4 3 3 3
3
Experimental Example 12
Experiments on Rats Receiving Treatments 24 h after the Onset of
Stroke with Compound Ie of the Present Invention at an Initial
Dosage of 5 .mu.mol/kg and 5 Subsequent Dosages of 2 .mu.mol/kg
Each
[0199] The efficacy was represented by neural function scores, and
a lower score indicates a higher efficacy. A 10% chloral hydrate
solution was injected intraperitoneally into male SD rats at a
dosage of 400 mg/kg body weight for anesthesia. A longitudinal open
incision was made at the center of the neck, and the right common
carotid artery trunk was dissected (about 3 cm in length). Carotid
external artery branches were each dissected and ligated at the
hyoid level, and the carotid internal artery was dissected at the
swollen part of the neck. The open incisions in the carotid
internal artery and the proximal end of the common carotid artery
were occluded respectively with noninvasive arterial clips, and the
distal end of the carotid external artery was ligated. A catheter
containing 0.5 mL thrombus suspension in normal saline was inserted
in the carotid external artery trunk. At the same time when the
clip on the carotid internal artery was released, the 0.5 mL
thrombus suspension in normal saline in the catheter slowly flew
from the carotid external artery to its proximal end, and then was
injected into the arteries in brain through the carotid internal
artery. Subsequently, the proximal end of the carotid artery was
ligated, the arterial clips on the carotid internal artery and the
common carotid artery were released, and blood flow was restored.
After the wound was stitched up, 20,000 IU penicillin was
intramuscularly injected for prevention from infection. After 24 h,
compound Ie in normal saline (at an initial dosage of 5 .mu.mol/kg,
n=12) was infused through the tail vein. Then, infusion of compound
Ie in normal saline (at a dosage of 2 .mu.mol/kg, n=12) through rat
tail vein was carried out once per day for 6 consecutive days,
observed for 7 days. The rats were compared to themselves each day,
and evaluated for the degree of damage in neural function by the
Zealonga method. A score of 0 indicated no sign of loss in neural
function, 1 indicated the front limbs on the undamaged side could
not stretch out, 2 indicated walking toward the undamaged side, 3
indicated tail-chasing walking in circles toward the undamaged
side, 4 indicated involuntary walking with disturbance of
consciousness, and 5 indicated death. The experimental results are
shown in Table 12.
[0200] The data in Table 12 demonstrated that efficacy was shown in
rats that received treatment 24 h after the onset of stroke with
one dose of 5 .mu.mol/kg compound Ie on day 1 and one dose of 2
mmol/kg compound Ie per day for the following 5 days. Among the 12
rats that received the treatment, three were dead, while 8 out of
the remaining 9 rats recovered to have no sign of loss in neural
function, and one had only the sign of slight loss in neural
function. Thus, continuous treatment with compound Ie showed
therapeutic effect on stroke beyond the golden treatment
window.
TABLE-US-00016 TABLE 12 Efficacy in rats receiving treatment with
compound Ie of the present invention 24 h after the onset of stroke
Daily neural function scores (Mean .+-. SD) Rat No. Day 1 Day 2 Day
3 Day 4 Day 5 Day 6 Day 7 1 3 2 2 1 0 0 0 2 3 2 1 1 1 0 0 3 2 1 1 0
0 0 0 4 3 2 2 1 1 0 0 5 5 6 2 1 1 1 1 0 0 7 3 3 3 2 1 1 1 8 3 4 2 1
1 0 0 9 5 10 3 3 1 1 1 1 1 11 5 0 0 0 0 0 0 12 2 1 1 1 0 0 0
Experimental Example 13
Experiments on Rats Receiving Treatments with 6 Successive
Administrations of 2 .mu.mol/kg Compound Ie of the Present
Invention 6 h after the Onset of Stroke
[0201] The efficacy was represented by neural function scores, and
a lower score indicates a higher efficacy. A 10% chloral hydrate
solution was injected intraperitoneally into male SD rats at a
dosage of 400 mg/kg body weight for anesthesia. A longitudinal open
incision was made at the center of the neck, and the right common
carotid artery trunk was dissected (about 3 cm in length). Carotid
external artery branches were each dissected and ligated at the
hyoid level, and the carotid internal artery was dissected at the
swollen part of the neck. The open incisions in the carotid
internal artery and the proximal end of the common carotid artery
were occluded respectively with noninvasive arterial clips, and the
distal end of the carotid external artery was ligated. A catheter
containing 0.5 mL thrombus suspension in normal saline was inserted
in the carotid external artery trunk. At the same time when the
clip on the carotid internal artery was released, the 0.5 mL
thrombus suspension in normal saline in the catheter slowly flew
from the carotid external artery to its proximal end, and then was
injected into the arteries in brain through the carotid internal
artery. Different from the previous experimental examples, the
thrombus clots used in this experimental example was a remarkably
solid thrombus suspension in normal saline prepared by using more
aged thrombus having been stored at RT for 24 h, instead of the
thrombus suspension in normal saline prepared by using thrombus
stored at -24.degree. C. Subsequently, the proximal end of the
carotid artery was ligated, the arterial clips on the carotid
internal artery and the common carotid artery were released, and
blood flow was restored. After the wound was stitched up, 20,000 IU
penicillin was intramuscularly injected for prevention from
infection. After 6 h, compound Ie in normal saline (at an initial
dosage of 5 .mu.mol/kg, n=12) was infused through the tail vein.
Then, infusion of compound Ie in normal saline (at a dosage of 2
.mu.mol/kg, n=12) through rat tail vein was carried out once per
day for 6 consecutive days, observed for 7 days. The rats were
compared to themselves each day, and evaluated for the degree of
damage in neural function by the Zealonga method. A score of 0
indicated no sign of loss in neural function, 1 indicated the front
limbs on the undamaged side could not stretch out, 2 indicated
walking toward the undamaged side, 3 indicated tail-chasing walking
in circles toward the undamaged side, 4 indicated involuntary
walking with disturbance of consciousness, and 5 indicated death.
The experimental results are shown in Table 13.
TABLE-US-00017 TABLE 13 Efficacy in rats receiving treatment with 2
.mu.mol/kg compound Ie of the present invention 6 h after the onset
of stroke Daily neural function scores (Mean .+-. SD) Rat No. Day 1
Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 1 4 3 2 1 1 1 1 2 3 2 1 1 0 0 0
3 3 3 3 3 1 1 1 4 3 3 1 1 1 1 1 5 5 6 3 3 1 1 1 1 1 7 5 8 3 3 1 1 1
1 1 9 5 10 5 11 5 12 3 5
[0202] The data in Table 13 demonstrated that, in rat models 6 h
after the onset of stroke induced by aged thrombus, efficacy was
shown after 6 successive treatments with one dose of 2 .mu.mol/kg
compound Ie per day for 6 consecutive days. Among the 12 rats that
received the treatment, six were dead, while 1 out of the remaining
6 rats recovered to have no sign of loss in neural function, and
five had only the sign of slight loss in neural function. Thus,
continuous treatment with compound Ie showed therapeutic effect on
old stroke.
[0203] It should be noted that, because compounds Ia to Il except
Ie in Experimental examples 1 to 7 achieved the effects in NO free
radical scavenging, euglobulin colt lysis, thrombolysis,
anti-thrombus action, and treatment in rats with stroke similar to
those of compound Ie, the other compounds of Ia to Il may achieve
the same therapeutic effects on old stroke as compound Ie did.
Experimental Example 14
Experiments on Nanostructures of Compounds Ia to Il of the Present
Invention at a Concentration of 1.times.10.sup.-6M,
1.times.10.sup.-9M and 1.times.10.sup.-12 M
[0204] Compounds Ia to Il according to the present invention were
prepared into 1.times.10.sup.-6 M, 1.times.10.sup.-9 M and
1.times.10.sup.-12 M solutions, respectively. 10 .mu.L solution was
taken and dropped onto a copper grid with a filter paper placed
underneath, air dried, and then observed under a transmission
electronic microscope (TEM) (JEOL, JEM-1230). Photographs were
taken so as to record the morphology and particle size.
[0205] 1. Test compound: compounds Ia to Il of the present
invention
[0206] 2. Test method: the test compound (Ia to Il) was prepared
into 1.times.10.sup.-6M, 1.times.10.sup.-9M and 1.times.10.sup.-12M
solutions with triple-distilled water, respectively. A small amount
(about 10 .mu.l) was taken and dropped onto the surface of a copper
grid with a filter paper placed underneath, air dried, and were
then observed under TEM (JEOL, JEM-1230) for the morphology and
particle size which were recorded in photographs.
[0207] 3. Test results: results are shown in FIGS. 25 to 36. FIG.
25 shows the nanostructures of compound Ia according to the present
invention in 1.times.10.sup.-6 M, 1.times.10.sup.-9 M and
1.times.10.sup.-12 M aqueous solutions, and the nanostructures of
Ia in the aqueous solutions are nanospheres having a diameter of
3.1 to 86.1 nm; FIG. 26 shows the nanostructures of compound Ib
according to the present invention in 1.times.10.sup.-6 M,
1.times.10.sup.-9 M and 1.times.10.sup.-12 M aqueous solutions, and
the nanostructures of Ib in the aqueous solutions are nanospheres
having a diameter of 4.3 to 297.9 nm; FIG. 27 shows the
nanostructures of compound Ic according to the present invention in
1.times.10.sup.-6 M, 1.times.10.sup.-9 M and 1.times.10.sup.-12 M
aqueous solutions, and the nanostructures of Ic in the aqueous
solutions are nanospheres having a diameter of 2.2 to 165.7 nm;
FIG. 28 shows the nanostructures of compound Id according to the
present invention in 1.times.10.sup.-6 M, 1.times.10.sup.-9 M and
1.times.10.sup.-12 M aqueous solutions, and the nanostructures of
Id in the aqueous solutions are nanospheres having a diameter of
16.2 to 201.2 nm; FIG. 29 shows the nanostructures of compound Ie
according to the present invention in 1.times.10.sup.-6 M,
1.times.10.sup.-9M and 1.times.10.sup.-12M aqueous solutions, and
the nanostructures of Ie in the aqueous solutions are nanospheres
having a diameter of 3.3 to 138.9 nm; FIG. 30 shows the
nanostructures of compound If according to the present invention in
1.times.10.sup.-6 M, 1.times.10.sup.-9 M and 1.times.10.sup.-12 M
aqueous solutions, and the nanostructures of If in the aqueous
solutions are nanospheres having a diameter of 3.6 to 82.4 nm; FIG.
31 shows the nanostructures of compound and the nanostructures of
Ig in the aqueous solutions are nanospheres having a diameter of
6.3 to 264.5 nm; FIG. 32 shows the nanostructures of compound Ih
according to the present invention in 1.times.10.sup.-6 M,
1.times.10.sup.-9 M and 1.times.10.sup.-12 M aqueous solutions, and
the nanostructures of Ih in the aqueous solutions are nanospheres
having a diameter of 5.1 to 149.8 nm; FIG. 33 shows the
nanostructures of compound Ii according to the present invention in
1.times.10.sup.-6 M, 1.times.10.sup.-9 M and 1.times.10.sup.-12 M
aqueous solutions, and the nanostructures of Ii in the aqueous
solutions are nanospheres having a diameter of 4.7 to 107.7 nm;
FIG. 34 shows the nanostructures of compound Ij according to the
present invention in 1.times.10.sup.-6 M, 1.times.10.sup.-9 M and
1.times.10.sup.-12 M aqueous solutions, and the nanostructures of
Ij in the aqueous solutions are nanospheres having a diameter of
9.1 to 73.7 nm; FIG. 35 shows the nanostructures of compound Ik
according to the present invention in 1.times.10.sup.-6 M,
1.times.10.sup.-9M and 1.times.10.sup.-12M aqueous solutions, and
the nanostructures of Ik in the aqueous solutions are nanospheres
having a diameter of 10.1 to 66.7 nm; FIG. 36 shows the
nanostructures of compound Il according to the present invention in
1.times.10.sup.-6 M, 1.times.10.sup.-9 M and 1.times.10.sup.-12 M
aqueous solutions, and the nanostructures of Il in the aqueous
solutions are nanospheres having a diameter of 6.1 to 153.3 nm.
Experimental Example 15
High Resolution FT-MS Experiments of Compounds Ia to Il of the
Present Invention at Concentrations of 1.times.10.sup.-6M,
1.times.10.sup.-9M and 1.times.10.sup.-12 M
[0208] Compounds Ia to Il were prepared into a 12.5 .mu.M solution
with triple-distilled water, and a 10 .mu.L sample was loaded onto
a solariX FT-ICR mass spectroscopy (Bruker Daltonik).
Intermolecular association status was observed and data was
acquired. The results are listed in Table 14 to 16.
TABLE-US-00018 TABLE 14 High resolution FT-MS data of dimers formed
by compounds Ia-Il of the present invention at three different
concentrations Concentration 1 .times. 10.sup.-6 M/ 1 .times.
10.sup.-9 M/ 1 .times. 10.sup.-12 M/ Compounds dimer dimer dimer Ia
2748.4580 2748.4580 2748.4580 Ib 2772.5308 2772.5308 2772.5308 Ic
2888.5308 2888.5308 2888.5308 Id 2720.4266 2720.4266 2720.4266 Ie
2744.4994 2744.4994 2744.4994 If 2840.4994 2840.4994 2840.4994 Ig
2606.3838 2606.3838 2606.3838 Ih 2630.4564 2630.4564 2630.4564 Ii
2726.4564 2726.4564 2726.4564 Ij 2294.1814 2294.1814 2294.1814 Ik
2318.2542 2318.2542 2318.2542 Il 2414.2542 2414.2542 2414.2542
TABLE-US-00019 TABLE 15 High resolution FT-MS data of trimers
formed by compounds Ia-Il of the present invention at three
different concentrations Concentration 1 .times. 10.sup.-6 M/ 1
.times. 10.sup.-9 M/ 1 .times. 10.sup.-12 M/ Compounds trimer
trimer trimer Ia 4122.1870 4122.1870 4122.1870 Ib 4158.2962
4158.2962 4158.2962 Ic 4332.2962 4332.2962 4332.2962 Id 4080.1399
4080.1399 4080.1399 Ie 4116.2491 4116.2491 4116.2491 If 4260.2491
4260.2491 4260.2491 Ig 3909.0757 3909.0757 3909.0757 Ih 3945.1846
3945.1846 3945.1846 Ii 4089.1846 4089.1846 4089.1846 Ij 3440.7721
3440.7721 3440.7721 Ik 3476.8813 3476.8813 3476.8813 Il 3620.8813
3620.8813 3620.8813
TABLE-US-00020 TABLE 16 High resolution FT-MS data of tetramers
formed by compounds Ia-Il of the present invention at three
different concentrations Concentration 1 .times. 10.sup.-6 M/ 1
.times. 10.sup.-9 M/ 1 .times. 10.sup.-12 M/ Compounds tetramer
tetramer tetramer Ia 5495.9160 5495.9160 5495.9160 Ib 5544.0616
5544.0616 5544.0616 Ic 5776.0616 5776.0616 5776.0616 Id 5439.8532
5439.8532 5439.8532 Ie 5487.9988 5487.9988 5487.9988 If 5679.9976
5679.9976 5679.9976 Ig 5211.7676 5211.7676 5211.7676 Ih 5259.9128
5259.9128 5259.9128 Ii 5451.9128 5451.9128 5451.9128 Ii 4587.3628
4587.3628 4587.3628 Ik 4635.5084 4635.5084 4635.5084 Il 4827.5084
4827.5084 4827.5084
[0209] Table 14 to 16 show the precise mass numbers measured by FT
High resolution MS. These mass numbers indicate that dimers,
trimers, and tetramers were all detected at three different
concentrations of compounds Ia-Il of the present invention.
Therefore, the compounds according to the present invention can
form dimers, trimers and tetramers in an aqueous solution at the
same time.
Experimental Example 16
High Resolution FT-MS Experiments of Compound Ie of the Present
Invention at Concentrations of 10.0 .mu.M, 1.0 .mu.M, 0.1 .mu.M and
0.01 .mu.M
[0210] For MS visualization, Compounds Ie was prepared into 10.0
.mu.M, 1.0 .mu.M, 0.1 .mu.M and 0.01 .mu.M solutions with
triple-distilled water, and a 10 .mu.L sample was loaded onto a
solariX FT-ICR mass spectroscopy (Bruker Daltonik). Intermolecular
association status was observed and data was acquired. The results
are shown in FIGS. 37 to 40. FIG. 37 is the high-resolution FT-MS
spectrum of compound Ie according to the present invention at a
concentration of 0.01 nM: 915.84146 is the triple-charged ion of
the dimer, 1030.32114 is the quadruple-charged ion of the trimer,
and 1099.00914 is the quintuple-charged ion of the tetramer; FIG.
38 is the high-resolution FT-MS spectrum of compound Ie according
to the present invention at a concentration of 0.1 .mu.M: 915.84124
is the triple-charged ion of the dimer, 1030.32208 is the
quadruple-charged ion of the trimer, and 1099.00829 is the
quintuple-charged ion of the tetramer; FIG. 39 is the
high-resolution FT-MS spectrum of compound Ie according to the
present invention at a concentration of 1 nM: 915.84095 is the
triple-charged ion of the dimer, 1030.32067 is the
quadruple-charged ion of the trimer, and 1099.00914 is the
quintuple-charged ion of the tetramer; FIG. 40 is the
high-resolution FT-MS spectrum of compound Ie according to the
present invention at a concentration of 10 nM: 915.84163 is the
triple-charged ion of the dimer, 1030.32067 is the
quadruple-charged ion of the trimer, and 1099.00914 is the
quintuple-charged ion of the tetramer.
[0211] The dimers, trimers and tetramers formed by the compounds of
the present invention further assembled into nanospheres having a
diameter of 2 to 300 nm. Among nanospheres of such sizes,
nanospheres having a diameter less than 100 nm was over 99%. It is
a well known fact in nanopharmacology that nanospheres having a
diameter of less than 100 nm are unlikely to be engulfed by
macrophages during transportation in blood and may readily cross
the capillary wall. These properties allow the compounds according
to the present invention to cross the blood-brain barrier. It is
the property of crossing the blood-brain barrier of the compounds
according to the present invention that enables the metabolic
products of the compounds according to the present invention to be
detectable in brain tissues in rats receiving treatment of
stroke.
Experimental Example 17
Experiments on High-Resolution FT-MS Monitoring the Metabolic
Products in Brain Tissues in Rats Treated with Compound Ie
According to the Present Invention
[0212] The entire rat brain was taken out and placed into a 50 mL
centrifuge tube, into which 10 mL 0.9% NaCl was added, and
homogenized to obtain a uniform suspension which was then
centrifuged at 3000 rpm for 10 min. 5 mL supernatant was added into
10 mL methanol and evenly mixed by shaking, and centrifuged at 3000
rpm for 10 min. The supernatant was concentrated under reduced
pressure until dry, followed by addition of 1 mL methanol, and
again centrifuged at 12000 rpm for 10 min. The resultant
supernatant was used for monitoring of the content of metabolic
products in brain tissues in rats treated with compound Ie.
[0213] High-resolution FT-MS experimental results showed two
metabolic products M1 and M2 in the brain. Among them, M1 had a
[M+1].sup.+ of 291.06971 and a molecular formula of
C.sub.15H.sub.19O.sub.5N.sub.2; and M2 had a [M+1].sup.+ of
307.04350 and a molecular formula of
C.sub.15H.sub.19O.sub.4N.sub.2. (MS conditions: loading: 10 .mu.L;
ionization mode: ES+; cone voltage: 30 V; mobile phase flow rate:
0.2 mL/min). According to the above data, the metabolic products M1
and M2 were assumed as the following compounds:
##STR00011##
TABLE-US-00021 M1 M2 Theoretical MW 291.1345 307.1249 Measured MW
291.0697 307.0435
[0214] This demonstrated that the compound Ie of the present
invention did indeed cross the blood-brain barrier, enabling the
effect of NO free radical scavenging, thrombolysis and the
anti-thrombus effect in brain.
* * * * *