Note: Descriptions are shown in the official language in which they were submitted.
CA 02457056 2004-02-10
PHARMACEUTICAL COMPOSITIONS COMPRISING POLYSACCHARIDE CONJUGATES FOR
INHIBITING THE METASTASIS OR PREVENTING THE RECURRENCE OF MALIGNANT TUMOR
TECHNICAL FIELD
The present invention relates to a pharmaceutical
composition for inhibiting the metastasis or preventing the
recurrence of a malignant tumor. More particularly, the
present invention relates to a pharmaceutical composition
for inhibiting the metastasis or preventing the recurrence
of a malignant tumor, which comprises as the active
ingredient a polysaccharide derivative comprising a
polysaccharide having a carboxyl group bound to an active
substance having an anti-tumor activity, for example, a
camptothecin derivative of the formula (I) or (II) as
mentioned below, via an amino acid or a peptide consisting
of 2 to 8 amino acids which are the same or different, or a
salt thereof.
BACKGROUND ART
Malignant tumors are one of the main causes of death
in developed countries, and the majority of malignant
tumor related deaths are due to metastasis into distant
organs or recurrence accompanied by metastasis to distant
CA 02457056 2004-02-10
2
organs after a topical therapy. The metastasis to distant
organs may be caused by hematogenous metastasis or
lymphogenous metastasis, and it is known that a patient
having lymphogenous metastasis has a high risk of
recurrence of a malignant tumor after topical therapy. The
main organs of recurrence are brain, lung, liver, and bone.
Especially, a digestive system tumor, for example,
colon cancer from which a large number of patients
suffer may often invade and spread to the liver, and
breast cancer and lung cancer as well often invade and
spread to the liver. Further, lymphoma and lymphatic
leukemia may spread mainly to the lymph system, and it has
been reported that the metastasis to liver was observed in
high rate by autopsy.
In order to inhibit the recurrence including the
metastasis to distant organs such a metastasis to the liver
and to prolong life, a chemotherapy, etc. is employed as a
supportive care after a topical therapy, but the
chemotherapy has a potent toxicity and cannot be used for
chronic administration. In addition, it has scarcely been
reported that life is more prolonged by
supportive care of chemotherapy than topical therapy
alone. For example, in the trials of post-surgery
chemotherapy for a patient who is the subject of surgery
of advanced gastric cancer, one of the cancers of the digestive
CA 02457056 2004-02-10
3
organs, clinical tests of various agents for anti-malignant
tumors have been tried, but any therapeutic method
exhibi t ing a remarkably better survival rate than surgery
alone has not yet been established.
Under these circumstances, it has been desired to find
a new agent effective in inhibiting recurrence or in
prolonging life after topical therapy, which is applicable
to the lymph node and the distant organs of metastasis with
few side effects, and is suitable for chronic
administration.
on the other hand, WO 94/19376, WO 97/46260, WO
97/38727, JP-A-10-72467 and JP-A-10-95802 disclose a
polysaccharide derivative comprising a polysaccharide bound
to an active substance having an anti-tumor activity via an
amino acid or a peptide.
However, these publications disclose the use of these
polysaccharides in the treatment of cancers by accumulating
at the tumor site and killing the tumor cells, but never
indicate activities of inhibiting metastasis or preventing
recurrence of a malignant tumor.
DISCLOSURE OF INVENTION
An object of the present invention is to provide a
novel pharmaceutical composition for inhibiting the
metastasis or preventing the recurrences of a malignant
CA 02457056 2007-05-29
4
tumor.
The present inventors have intensively studied, and
have found that a polysaccharide derivative comprising a
polysaccharide having a carboxyl group bound to an active
substance having an anti-tumor activity via an amino acid or
a peptide exhibits an excellent effect in the inhibition of
metastasis and/or prevention of recurrence of a malignant
tumor, and have accomplished the present invention. That
is, the present invention relates to pharmaceutical
composition for inhibiting the metastasis or preventing the
recurrence of a malignant tumor, which comprises as the
active ingredient a polysaccharide derivative comprising a
polysaccharide having a carboxyl group bound to an active
substance having an anti-tumor activity via an amino acid or
a peptide consisting of 2 to 8 amino acids which are the
same or different, or a salt thereof.
In accordance with one aspect of the present invention
there is provided a pharmaceutical composition for
inhibition of metastasis or prevention of recurrence of a
malignant tumor, which comprises as the active ingredient a
polysaccharide derivative comprising a polysaccharide
having a carboxyl group bound to an active substance having
an anti-tumor activity via an amino acid or a peptide
consisting of 2 to 8 amino acids which are the same or
different, or a salt thereof, together with at least one
pharmaceutically acceptable excipient, diluent, or carrier.
CA 02457056 2007-05-29
4a
In accordance with another aspect of the present
invention there is provided use of a polysaccharide
derivative comprising a polysaccharide having a carboxyl
group bound to an active substance having an anti-tumor
activity via an amino acid or a peptide consisting of 2 to
8 amino acids which are the same or different, or a salt
thereof for inhibition of metastasis or prevention of
recurrence of a malignant tumor.
In accordance with yet another aspect of the present
invention there is provided use of a polysaccharide
derivative comprising a polysaccharide having a carboxyl
group bound to an active substance having an anti-tumor
activity via an amino acid or a peptide consisting of 2 to
8 amino acids which are the same or different, or a salt
thereof for the manufacture of a medicament for inhibition
of metastasis or prevention of recurrence of a malignant
tumor.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig 1 shows the lapsed days after implantation of
tumor and the number of survived animals in M 5076 liver
metastatic models.
BEST MODE FOR CARRYING OUT THE INVENTION
The polysaccharide having a carboxyl group of the
CA 02457056 2004-02-10
present invention includes the same ones as those disclosed
in the above mentioned WO 94/19376 and WO 97/46260, and
includes a polysaccharide having originally carboxyl groups
in the structure thereof (e.g., hyaluronic acid, pectic
5 acid, alginic acid, chondroitin, heparin, etc.), and
polysaccharides having originally no carboxyl group (e.g.,
pullulan, dextran, mannan, chitin, mannoglucan, chitosan,
etc.) but being introduced thereto carboxyl groups, and
polysaccharides having originally no carboxyl group in the
structure thereof but being introduced thereto carboxyl
groups after polyalcohol formation (e.g., polysaccharide
polyalcohol having a carboxyl group).
The polysaccharide having originally no carboxyl group
but being introduced thereto a carboxyl group means one
that prepared by substituting a hydrogen atom forapart
oraliofthe hydroxyl groups of polysaccharides having
originally no carboxyl group with a carboxy-C1_, alkyl group.
In the present invention, the polysaccharide having a
polysaccharide includes one that is prepared by treating a
polysaccharide originally having no carboxyl group=with a
reducing agent, and then followed by substituting a
hydrogen atom fora part or all of hydroxyl groups of the
resultant compound with a carboxy-C1_, alkyl group.
The polysaccharide polyalcohol having a carboxyl group
includes, for example, a carboxy-C1_, alkyl-polysaccharide
CA 02457056 2004-02-10
6
polyalcohol which is prepared by treating a polysaccharide
originally having no carboxyl group successively with
sodium periodate and sodium borohydride by the method
disclosed.in WO 97/46260 to give a polysaccharide
polyalcohol, which is further treated with a halogenated
C1_4 alkylcarboxylic acid.
The alkyl moiety of the carboxyl-C,_, alkyl group which
substitutes a hydrogen atom of the hydroxyl groups of the
above polysaccharide (including a polysaccharide
polyalcohol) may be either a straight chain alkyl group or
a branched chain alkyl group.
A preferred carboxy-C,_, alkyl group is, for example,
carboxymethyl group, 1-carboxyethyl group, 3-carboxypropyl
group, 1-methyl-3-carboxypropyl group, 2-methyl-3-carboxy-
propyl group, 4-carboxybutyl group, etc., and carboxymethyl
group is more preferable.
In the present invention, the polysaccharide having a
carboxyl group is preferably carboxy-C,_, alkyldextran or
carboxy-C1_, alkyldextran polyalcohol, and carboxyl-C,_,
alkyldextran is especially preferred.
The degree of polyalcohol formation (by the successive
oxidation with sodium periodate and reduction with sodium
borohydride) in the step of preparing the carboxy-C1_4
alkyl-polysaccharide polyalcohol as mentioned above is not
specified, but the intermediate polysaccharide polyalcohol
CA 02457056 2004-02-10
7
is preferably one being obtained by treating a
polysaccharide under possible conditions for substantially
almost completely forming a polyalcohol.
Moreover, in the present invention, the polysaccharide
having a carboxyl group is preferably carboxymethylated
dextran or carboxymethylated dextran polyalcohol, and among
these polysaccharides, particularly dextran having an
average molecular weight of 20,000 to 500,000 is more
preferable, and dextran having an average molecular weight
of 50,000 to 350,000 is most preferable (said average
molecular weight being determined by gel permeation
chromatography (GPC) method, Shinseikagaku, Jikken Koza,
vol. 20, p. 7, Tokyo-Kagaku-Dojin, November 5, 1991).
When introducing a carboxylalkyl group into
polysaccharides, the degree of the introduction thereof is
expressed by "degree of substitution" which is defined by the
number of carboxylalkyl groups (including groups of peptide
chain being introduced by these groups) per sugar residue.
That is expressed by the following equation.
Number of carboxyalkyl groups
Degree of in the molecule
Substitution Total number of
sugar residues in the molecule
When the carboxylalkyl group is a carboxymethyl group,
the degree of substitution is occasionally expressed by the
degree of carboxymethylation (CM-degree).
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8
When the polysaccharide is dextran, the de.gree of
substitution thereof is preferably in the range of 0.3 to
0.8. When the polysaccharide is dextran polyalcohol, the
degree of substitution is preferably in the range of 0.3 to
0.5.
The amino acid or peptide of the present invention
plays a role of spacer existing between a polysaccharide
having a carboxyl group and an active substance having an
anti-tumor activity, and the amino acid or amino acid
forming said peptide includes both natural amino acids and
synthetic amino acids (including D-amino acids, L-amino
acids, a mixture thereof), and also includes either neutral
amino acids, basic amino acids or acidic amino acids.
Moreover, the amino acid of the present invention may be
not only a-amino acid but also R-amino acids, y-amino acids,
e-amino acids, etc.
Examples of the amino acids are glycine, a-alanine, R-
alanine, valine, leucine, isoleucine, serine, threonine,
systeine, methionine, aspartic acid, glutamic acid, lysine,
citrulline, arginine, phenylalanine, tyrosine, histidine,
tryptophan, proline, hydroxyproline, y-aminobutyric acid,
E-aminocaproic acid, etc.
. The peptide of the present invention includes ones
consisting of 2 to 8 amino acids, preferably 2 to 5 amino
acids, which are the same or different. Examples of the
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9
peptides are glycyl-glycyl-L- or D-phenylalanyl-glycine,
glycyl-glycine, glycyl-glycyl-glycine, glycyl-glycyl-
glycyl-glycine, glycyl-glycyl-glycyl-glycyl-glycine, L- or
D-phenylalanyl-glycine, L- or D-tyrosyl-glycine, L- or D-
leucyl-glycine, L- or D-phenylalanyl-citrulline and L- or
D-valyl-citrullin (the N-terminus of these peptides is
introduced onto the carboxyl group of a polysaccharide).
Among these peptides, glycyl-glycyl-L- or D-phenyl-
alanyl-glycine, glycyl-glycine, glycyl-glycyl-glycine,
glycyl-glycyl-glycyl-glycine, glycyl-glycyl-glycyl-glycyl-
glycine, and L- or D-phenylalanyl-glycine are preferable.
The active substance having an anti-tumor activity of
the present invention may include various compounds
known as an anti-tumor agent, and may be either cytotoxic
agents or cytostatic agents. The cytotoxic agent is
preferably camptothecin derivatives and taxane derivatives,
and the cytostatic agent is preferably angiogenesis
inhibitors, EGF receptor inhibitors. More preferably, the
cytotoxic agent is camptothecin derivatives, and the
cytostatic agent is angiogenesis inhibitors.
Examples of camptothecin derivatives are compounds
disclosed in JP-A-10-72467 of the formula (I):
CA 02457056 2004-02-10
R' O
X'-AIk-O N ~ ~ (I)
N
HsC2 OH
wherein R' is a substituted or unsubstituted lower alkyl
group, X1 is a group of the formula: -NHR2 (RZ is a hydrogen
atom or a lowe.r alkyl group) and Alk is a straight chain or
5 branched chain C1_6 alkylene group having optionally an
oxygen atom in the chain thereof. Among them, a preferred
compound is 10-(3'-aminopropyloxy)-7-ethyl-(20S)-
camptothecin.
Other examples of camptothecin derivatives are
10 compounds disclosed in JP-A-10-95802 of the formula (II):
R3 R2 0
R4
~ O (II)
R5 6 N O
R H5C2 OH
wherein two groups of R 2 to R6 being adjacent each other
combine to form a lower alkylene group, and one of the
carbon atoms of said lower alkylene group is substituted by
an amino group, and the remaining three groups of R2 to R6
are a hydrogen atom, a lower alkyl group or a halogen atom.
Among them, a preferable compound is (iS, 9S) -1-amino-9-
ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-
benzo [de]pyrano [31,41 : 6, 7] indolizino [1, 2-b] quinoline-
CA 02457056 2004-02-10
11
10, 13 (9H, 15H) -dione, etc.
Examples of taxane derivatives are Taxo1T"', TaxotereTM
,
13- [ (2 ' R, 3' R) -3' N-t-butyloxycarbonyl-3' -cyclopropyl] -10-
deacetyl-baccatin III, etc.
In the active ingredient of the present invention, the
ratio of the polysaccharide and the active substance having
an anti-tumor activity may be selected according to the
kinds of the polysaccharide to be used, but when the
polysaccharide is dextran or dextran polyalcohol, then the
content of the active substance having an anti-tumor
activity is preferably in the range of 0.1 to 20 % by
weight, more preferably in the range of 2 to 10 % by weight,
based on the total weight of the active ingredient.
Among the active ingredients of the present invention,
preferable ones are polysaccharide derivatives or a salt
thereof wherein an amino acid or a peptide consisting of 2
to 8 amino acids which are the same or different are
introduced into a part or all of the carboxyl groups of the
polysaccharide having a carboxyl group through an acid-
amide bond, and the remaining part or all of the amino
groups or carboxyl groups which do not participate in the
binding to the carboxyl groups of the above peptide are
bound to the carboxyl groups, amino groups or hydroxyl
groups of the active substance having an anti-tumor
activity through an acid-amide bond or ester bond.
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12
An especially preferable active ingredient is a
polysaccharide derivative, wherein the polysaccharide
having an carboxyl group is carboxymethylated dextran, the
active substance having an anti-tumor activity is 10-(3'-
aminopropyloxy)-7-ethyl-(20S)-camptothecin, and the peptide
is glycyl-glycyl-glycine, or a salt thereof. Especially
preferred is a polysaccharide derivative wherein the
polysaccharide having a carboxyl group is carboxymethylated
dextran having an average molecular weight of 60,000 to
200,000, and the degree of carboxymethylation thereof is in
the range of 0.3 to 0.8, or a salt thereof.
Another preferred active ingredient is a polysaccharide
derivative wherein the polysaccharide having a carboxyl
group is a carboxy-C1_, alkyldextran polyalcohol, the active
substance having an anti-tumor activity is (1S, 9S)-1-
amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-
1H,12H-benzo[de]pyrano[3',4':6,7] indolizino [1,2-b]-
quinoline-10,13(9H,15H)-dione, and the peptide is glycyl-
glycyl-L- or D-phenylalanyl-glycine, or a salt thereof, and
especially preferred is a polysaccharide derivative
or a salt thereof wherein the polysaccharide having a
carboxyl group is carboxy-C,_, alkyldextran polyalcohol
having an average molecular weight of 200,000 to 400,000,
and the degree of the substitution thereof is in the range
of 0.3 to 0.5.
CA 02457056 2004-02-10
13
The polysaccharide derivative or a salt thereof of the.
active ingredient of the present invention may be prepared
according to the methods disclosed in WO 94/19376, WO
97/46260, WO 97/38727, JP-A-10-72467, and JP-A-10-95802.
The pharmaceutical composition of the present
invention may highly accumulate at the site such as lymph
node or the liver to which cancers may spread, can release
an active substance at an appropriate rate so that the
active substance hardly affects the normal cells and
suppressively acts on the growth of tumor cells, and hence,
the pharmaceutical composition of the present invention is
useful in the inhibition of metastasis or prevention of
reoccurrence of a malignant tumor. Especially, the
pharmaceutical composition of the present invention is
useful in the inhibition of lymph node metastasis or liver
metastasis, particularly useful in the inhibition of lymph
node metastasis. Further, among lymph node metastasis, the
present pharmaceutical composition is useful in the
inhibition of metastasis in lymph nodes from the colon, or
metastasis in lymph nodes from the lung.
In addition, the present pharmaceutical composition
may exhibit its effects not only before the onset of
metastasis but also after the onset of metastasis.
Therefore, the present pharmaceutical composition is also
useful in the inhibition of metastasis or prevention of
CA 02457056 2004-02-10
14
reoccurrence of a malignant tumor after a topical therapy
(e.g., surgery, radiation therapy, thermotherapy,
cryothe rapy, laser burning therapy, etc.). Moreover, the
present pharmaceutical composition is also suitable for
repetitive dosing over a long time, and can be employed
together with a topical therapy.
The present pharmaceutical composition is preferably
administered parenterally (e.g., intravenous injection),
and is usually administered in the form of a liquid
preparation such as solution, suspension, emulsion, etc.
The present pharmaceutical composition is preferably
formulated in the form of an injection or drip infusion by
using distilled water for injection, physiological saline
solution, aqueous glucose solution.
The dosage of the present pharmaceutical composition
may vary according to the administration methods, age,
weight or conditions of the patients, etc., but it is
usually in the range of 0.002 to 50 mg/kg, more preferably
in the range of 0.01 to 5 mg/kg, in single dose, converted
into an amount of the active substance.
In the present specification, the lower alkyl group
and the lower alkylene group may be those having 1 to 6
carbon atoms, preferably those having 1 to 4 carbon atoms,
and the halogen atom is fluorine atom, chlorine atom,
bromine atom, iodine atom, etc.
CA 02457056 2004-02-10
EXPERIMENTS
Experiment 1 (M 5076 liver metastatic models)
One million of M 5076 cells (mouse ovarian sarcoma
cells) were implanted into BDF1 male mice (5-weeks old, 8
5 animals per group) at the tail vein. A test compound
(Compound A; the compound obtained in Preparation 1 as
described below and lrinotecanTM (CPT-11) ) was dissolved in a
physiological saline solution, and each amount as indicated
in Table 1 as mentioned below was administered
10 intravenously to the mice on the 4th, 8th and 12th day
after the implantation, and the mice were observed for 120
days after the implantation of tumor. In the control group
(untreated with test compound), only a physiological saline
solution was administered. The survival time (days) was
15 measured in both the test compound-treated groups and the
control group, and the prolongation rate of survival was
calculated according to the following equation. The
results are shown in Table 1 and Fig. 1.
CA 02457056 2004-02-10
16
Survival days in the test
Prolongation rate _ compound-treated group
of survival - 1 X 100
1 Survival days in
the control group
Table 1
Prolon-
Dose Survival Standard gation
(mg/kg) days error rate of
survival
M
Control 15.00 1.24 -
Compound A 12.5 37.57 5.27 150.5
25 43.13 5.98 187.5.
50 48.71 5.33 224.8
Irinotecan 80 22.50 0.5 50.0
As is shown in Table 1, the compound obtained in
Preparation 1 as mentioned below (Compound A) exhibited
excellent activity of prolonging lifetime in M5076 liver
metastatic models. Meanwhile, Irinotecan is not known as
an agent for inhibiting the metastasis or preventing the
reoccurrence of a malignant tumor, but it was merely tested
as a drug of camptothecin derivatives.
Experiment 2 (HT-29 metastatic models)
A segment (2 mmZ) of HT-29 cells (human colon cancer)
was implanted into the vermiform appendix of 100NCr nu/nu
female mice (5 to 6 weeks old, 10 animals per group). A
test compound (Compound A; the compound obtained in
Preparation 1 as mentioned below, Compound B; the compound
obtained in Preparation 4 as mentioned below, and
CA 02457056 2004-02-10
WO 03/015826 PCT/JP02/08309
17
Irinotechan (CPT-11)) was dissolved in a physiological
saline solution, and each amount as indicated in Table 2 as
mentioned below was administered intravenously to the mice
on the 15th, 19th, 23rd and 25th day after the implantation
of tumor. On the other hand, in the control group
(untreated with test compound), only a physiological saline
solution was administered. The presence or absence of the
metastasis of each organ was checked on the 84th day after
the implantation of tumor. The results are shown in the
following Table 2.
CA 02457056 2004-02-10
18
H O r-I ao
44 4=- rn # o O O m o cr) u) 0
41 ~
O =.i =rl ~ ~
m a o O o O o ~ ~ j1
ca v v v
~ ai ~
v
N
~
C) N OD kD 0 t0 [- cn cn
z o~ ~ o
'4 x a
c'.' O +
o 0 0 O o 0 0 O A k
x
=r. LO
s~#
~ ~~i rn
4-1 ro a o -
0 01 * 0 0 1-1 rl O O N
O ~ O 0 rl rl C) r 1 () a~ r1 %
0 s.C
4-) rt =.i '.,
U 'p
ri co co .-1 CA co y 4) ~ N u) O u7 N If) u) O
= = = ' O N =-i
IL O C) rl C) C) C) 11
O '~ =~ v ~ O
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al U
#
t-4 O rl N r-i O ri .-1 N f'') a 04 =~ 4J w~Q~
t01 ~U /1 Ln N
tT ~t l~ I
0 I .-i =-i
N * O O O O C) O O H .C
~,
O ~ 4 .k . . . . . . .
to a o
A P ~ rt >I v- ,N
E o o
a ~ O C) O 0 0 r~I 0
N ~-( to
> O-rz
.-1 .-i .-1 O co =~ .-1 61 I w
CD 0 o cn O (Y) un o a~ ro i._ ==
o a, o o ~ O o O ~~ ~m ~ d'
v v v "
=o 0 o i ~.~
U C) 0 LL * H ~ ~ ~ O ~
H O ~ I OD lfl O kO [- m ~
u~ E C+' k
.. 4 m tn ..~ C >1
41 y4 N +%~ ~4 m y N
a) ua 'U O O o O o O O O o ~ ro ro04 ~
p 41 'o .a a) 0
~ 4l ~ G1 D ~
C ~d 41 tS O Cp
al ~ G U U
C y =.i I
Ei +J ~ ro b
a T1 ~ C3 't3 = "C pl U X U x '~ H~ d~ OO O'.C
~+ C~' CX C tT C~ CX N~ N~ N Zw ~Ca A.-i
~'k a O C.Y o 34 ::% -- 41 CT 41 tT
0 n. n. a a ~ a ~ ~. ~ 5 a E
~ ~ E ~ ~ ~ ~ ~ =.~ o .~ O V .. *
V O U O V O V~ 0 O~ v H N .. ##
('j '=.i U7 V N .. #= # # #
# i~ # # #
CA 02457056 2004-02-10
WO 03/015826 PCT/JP02/08309
19
Experiment 3 (HT-29 metastatic models)
A segment (2 mm2) of HT-29 cells (human colon cancer)
was implanted into the vermiform appendix of 100NCr nu/nu
female mice (5 to 6 weeks old, 10 animals per group).
Since the lymph node metastasis was observed on the 49th
day after the implantation of tumor, a test compound
(Compound A; the compound obtained in Preparation 1 as
mentioned below, and Irinotechan (CPT-11)) was dissolved in
a physiological saline solution, and each amount as
indicated in Table 3 as mentioned below was administered
intravenously to the mice on the 51st, 55th, 59th, and 63rd
day after the implantation of tumor. On the other hand, in
the control group (untreated with test compound), only a
physiological saline solution was administered. The
presence or absence of the metastasis of each organ was
checked on the 84th day after the implantation of tumor.
The results are shown in the following Table 3.
CA 02457056 2004-02-10
WO 03/015826 PCT/JP02/08309
1..~ fA ic O 0 0
'~ -ri _4 ~ . . . i +J
0 m a, O u
0
ro v v x
cn +~
r0 r0 s4
z-~ ~ N cn ao rn U
ro
-~
w
0 O o >1
* * . . . i
A+ r i
O ~ 0 U
~
.7-J M 4
O ~ i~ 0 O
O H cV rl r-I r=I U a
O ~
+-) ro
.O U
ri '--I r-I a)
* N N N ~ H
(15
4.1 O
a O O O 0 =ri N
~J U ro ~
U r-I
rn
*
H 0 0 0 (V) -~
U N
~ 0
}a ro I
O I
# 0 0 0 ~ H >i
ch * . . . ~ 4-, x
.~ ~ r+ ro
y4 a z~ o
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04
0 0 H . I ~ -~ o 04
y~ U -rl
r-I rl a) .-1 ~
o 0 0 ~ u ~'
o a o o
v v
-~ ~
R~ U 4-)
rl
H 0 N 00 Ol
U 3-i 4-)
F_:
4-) ro
~ .~ (L)
O b H 4-4 ~ O o 0 0 fa ro ru p
o -ro H ~ ca >
v) U) 9 U
+ ~d U)
T3 N
FZ~ ~ ~.; .- N
~' 0 ~a~r9 A
0 4-) O,
aa~o rg 4J
O o O O~~ U ..
U ~ C~ N H '' .. **
== -k i~ ~
* ~ ~ ~
CA 02457056 2004-02-10
21
Experiment 4 (H460 metastatic models)
A segment (2 mmZ) of H460 cells (human lung cancer)
was implanted into the left lung of 100NCr nu/nu female
mice (5 to 6 weeks old, 10 animals per group). Since the
metastasis was observed on the 14th day after the
implantation of a tumor in another control group, a test
compound (Compound A; the compound obtained in Preparation
1 as mentioned below, Compound B; the compound obtained in
Preparation 4 as mentioned below, and Irinotechan (CPT-11))
was dissolved in a physiological saline solution, and each
amount as indicated in Table 4 as mentioned below was
administered intravenously to the mice on the 14th, 18th,
22nd and 26th day after the implantation of tumor. On the
other hand, in the control group (untreated with test
compound), only a physiological saline solution was
administered. The presence or absence of the metastasis of
each organ was checked on the 36th day after the
implantation of tumor. The results are shown in the
following Table 4.
CA 02457056 2004-02-10
22
}.t
LO
f~ * ~ 1 cM (V t0 0 N N l0 >1
44 -W fn O N O O O O O O a) 4J
O I . I , . . .
3 m ' o 0 0 0 0 0 0 o
~4 rt v
4) ,-Ni v'~i ~ o
1 4J
I
'~ 2 ~ ~--I m ri N N .-i ~--I N N ..I . G'~ 0
>1 N
ro A G >, LO
.~ .I C = -I
.
cr o~ ~= C O
LO LO LO C) o u) o u) ~ ~=0 ~
* . = 1 V ~ ~ ~
o 0. 0 .1 r+ o r+ o
a >,
0
41
7 .O U 'C ~
(,
-
O N O rl .ri 0 1-1 0 CN ro En "ri
O N r--1
1 O
O O ~
U =1 O O
f3. .[
K O O O O O O O 0 1 0 +) 44 ,Q
* . . . . . . . O O 1 1
p~ .-i ~--I .--1 ~i .-i .--I .--1 =--I -4 I LP) N
-.
1 1J ~C O
~ a * ro O d .C
,-I H O O O O 0 O O O O ~ ~a% N
A ~ 4.) 0. 1
0
ro
F' 0 o W) vn b
C) 0 0 0 0 0 0 o
E ~+
o w o 0 0 0 0 0 0 o ro~ toI'. .
v v v v ~~
~ V 0 1 .
"1 =rl O tn ~
H 1 CV 1-1 N C) N U i-1 rl v
'--
a ~ ~ =%-I ~-Ui O r0
r0
41
U fa 1-1
+v "H.14a
s+ ,~ ~ 'o m ~
y.a o O o 0 0 0 0 0 o ro ro 41
~ 4 . ~ .1 r-i N .p 0 H 0 0
o .-
ro a) o > m 0 U U -0I
= õ ro ~ a~ a~ x
a y4j ro ro~
roAd U~ ,~ F VI Ol tA
p~ 1y ~ O~ L7 ~ O= i3 ~ U 0 H N o O x
r1 v~ ~ x x a a~ tr, ~ x d~ d~ ~4 4-1 ca ca .-1 ~ x a a x 3 x ~:j~ tr 4-) m 0
o~ o Or O~ ~
t9 r E R E ~ -+ o ~ o o .. *.
O 0 O 0 O 0 0 0 in H v H m U **
U = == ++
UC) U N U,~ Uto U uo
N .. ic # *
+ # # k
CA 02457056 2004-02-10
WO 03/015826 PCT/JP02/08309
23
PREPARATIONS
Preparation 1
Preparation of CM-dextran-7-ethyl-10-[3'-(glycyl-glycyl-
glycylamino)propyloxy]-(20S)-camptothecin:
O
CM-Dextran-(GIy)3-NH-(CH2)3-O N O
N O
H5C2 OH
(CM-Dextran means carboxymethyldextran, hereinafter, the
s ame )
(1) 10-(3'-P,minopropyloxy)-7-ethyl-(20S)-camptothecin
hydrochloride (500 mg) was dissolved in acetonitrile (25
ml), and thereto were successively added t-butoxycarbonyl
glycyl-glycyl-glycine (345 mg), N-methylmorpholine (121 mg),
N-hydroxybenzotriazole (161 mg) and 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide hydrochloride (228 mg), and the
mixture was stirred overnight. The precipitated product
was collected by filtration, purified by silica gel column
chromatography to give pale yellow foamy powder, which was
recrystallized from n-propanol to give 7-ethyl-10-[3'-(t-
butoxycarbonyl-glycyl-glycyl-glycylamino)propyloxy]-(20S)-
camptothecin (663 mg) as colorless crystals.
M.p.: 157-159 C.
(2) 7-Ethyl-10-[3'-(t-butoxycarbonyl-glycyl-glycyl-
glycylamino)propyloxy]-(20S)-camptothecin (3.86 g) was
CA 02457056 2004-02-10
24
emulsified in purified water (64 ml), and thereto was added
6N aqueous hydrochloric acid solution (32 ml), and the
mixture was reacted at room temperature under stirring for
2 hours. The solvent was concentrated, and thereto was
added n-propanol to precipitate a powdery product. The
resulting powdery product was collected by filtration, and
recrystallized from aqueous n-propanol to give 7-ethyl-l0-
[ 3' - (glycyl-glycyl-glycylamino) propyloxy] - (20S ) -
camptothecin hydrochloride (2.56 g) as yellow crystals.
(3) CM-Dextran sodium salt (CM-degree = 0.44, 50 g) was
dissolved in water (2.5 liters), and the pH value thereof
was adjusted to pH 5.0 with 0.2N aqueous hydrochloric acid
solution under stirring at 15 C, and thereto was added 7-
ethyl-l0-[3'-(glycyl-glycyl-glycylamino)propyloxy]-(20S)-
camptothecin hydrochloride (4.01 g). To the mixture was
added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (50 g), during which the pH value of the
reaction solution was kept at 5.0-5.5 with 0.2N
hydrochloric acid. The mixture was reacted at 15 C under
stirring for one hour, arid diluted to a total volume of 10
liters with purified water. While the pH value was kept at
over pH 4.0, the low molecule fractions were removed
using an ultrafiltration module (ACP-1010T"', manufactured by
Asahi Kasei Industries, Ltd.), and the pH value thereof was
adjusted to pH 8 with 0.1N aqueous sodium hydroxide
CA 02457056 2004-02-10
sol.ution, and then subjected to ion-exchange resin MSC-ITM
(Na-type, manufactured by Dowex). The fractions containing
the desired compounds are concentrated, and filtered
through a filter (0.45 }un) . The resultant product was mixed with
5 ethanol (10 liters) with stirring, and thereto was added
dropwise 3M brine (40 ml) under stirring. The resulting
precipitates were collected by filtration, and dissolved in
purified water (21 liters). The pH value of the solution
was adjusted to pH 4.0 with 0.2N aqueous hydrochloric acid
10 solution, and subjected again to ultrafiltration during
which the pH value was kept at pH 4Ø The solvent was
concentrated toa total volume of 1.5 liter, and filtered
through a filter (0.45 Iim) . The resultant product was mixed with
ethanol (9 liters), and thereto was added dropwise 3M brine
15 (35 ml) under stirring. The resulting precipitates were
collected by filtration, and washed successively with
ethanol and acetone, concentrated under reduced pressure to
give the desired compound (54.9 g)asa pale yellow powder.
The content as 10-(3'-aminopropyloxy)-7-ethyl-(20S)-
20 camptothecin hydrochloride was confirmed as 4.2 % by
absorption at 367.5 nm. According to the analysis by GPC
(gel permeation chromatography), the average molecular
weight of the desired product was 121 kDa, and the degree
of distribution (Mw/Mn) was 1.47.
CA 02457056 2004-02-10
26
Preparation 2
Preparation of CM-dextran-13-[(2'R,3'S)-3'-N-tert-
butoxycarbonyl-3'-phenyl-2'-O-L-phenylalanyl-glycyl-
isoserinyl]-10-deacyl-baccatin III:
HO O OH
ONHBoc
= O
CM-Dextran-Phe-GIy-O 0 OH Ac0
Bz0
(Bz is benzoyl group, hereinafter, the same)
CM-Dextran(2008 mg, CM-degree: 0.47, the average
molecular weight: 170 kDa) was dissolved with stirring in
purified water (90 ml), and thereto were added 13-
[(2'R,3'S)-3'-N-tert-butoxycarbonyl-3'-phenyl-2'-O-L-
phenylalanyl-glycyl-isoserinyl]-10-deacyl-baccatin III
mesylate (119 mg) and dimethylformamide (90-m1), and the
mixture was stirred so as to dissolve. To the mixture was
added with stirring 2-ethoxy-1(2H)-quinolinecarboxylic acid
(4.0 g), and the mixture was stirred at room temperature
overnight. To the reaction solution was added ethanol (720
ml) with stirring, and thereto was further added dropwise
3M brine (1.8 ml) under stirring. The precipitates were
collected by centrifugation, and dissolved in water (200
ml), and the pH value of the solution was adjusted to pH 7
with 0.2N aqueous sodium hydroxide solution. The solution
was poured into ethanol (800 ml) with stirring, and thereto
CA 02457056 2004-02-10
27
was added dropwise 3M brine (4 ml) with stirring.. The
resulting precipitates were collected by centrifugation,
and purified in the same manner as in Preparation 1-(3) to
give the desired compound (600 mg) as a white powder.
The content of the active substance: 2.4 % (W method, (X _
276 nm) )
Preparation 3
Preparation of CM-dextran-2'-O-phenylalanyl-glycyl-taxol:
AcO O OH
1NH-Bz
~ - O
CM-Dextran-Phe-GIy-O O OH Ac0
Bz0
CM-Dextran (1.294g, CM-degree: 0.47, the average
molecular weight: 170 kDa) was dissolved with stirring in
purified water (70 ml), and thereto were added 2'-O-phenyl-
alanyl-glycyltaxol mesylate (77 mg) and dimethylformamide
(70 ml), and the mixture was further stirred so as to
dissolve. 2-Ethoxy-1(2H)-quinolinecarboxylic acid (2.59 g)
was added to the mixture under stirring, and the mixture
was reacted with stirring overnight. The reaction solution
was added to ethanol (700 ml) under stirring, and thereto
was added dropwise 3M brine (1.4 ml) under stirring. The
precipitates were collected by centrifugation, and
dissolved in water (240 ml), and mixed with ethanol (1200
CA 02457056 2004-02-10
28
ml) under stirring. 3M Brine (4.8 ml) was added drowpise
to the mixture under stirring for precipitation. In the
same manner, the precipitation was further repeated three
times to give the desired product (746 mg) as a white powder.
The content of the active substance: 4.8 % (W method (A =
273 nm) )
Preparation 4
Preparation of carboxymethyldextran-polyalcohol-(1S,9S)-1-
(glycyl-glycyl-L-phenylalanyl-glycylamino)-9-ethyl-5-
fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]-
pyrano [3' , 4' : 6, 7] indolizino [1, 2-b] quinoline-10, 13 (9H, 15H) -
dione:
(1) Preparation of (IS,9S)-1-(t-butoxycarbonyl-glycyl-
glycyl-L-phenylalanyl-glycylamino)-9-ethyl-5-fluoro-2,3-
dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano-
[3', 4' : 6, 7] indolizino [1,2-b]quinoline-10, 13 (9H, 15H)-dione:
Boc-Gly-Gly-Phe-Gty
1
.,\NH 0
Me N O
F N~ O
.~' H5Cz OH
To a solution of (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-
dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano-
[3', 4' : 6, 7] indolizino [1, 2-b] quinoline-10, 13 (9H, 15H)-dione
hydrochloride (167 mg; 0.354 mmol), t-butoxycarbonyl-
CA 02457056 2004-02-10
29
glycyl-glycyl-L-phenylalanyl-glycine (463 mg; 1.06 mmol)
and 1-hydroxybenzotriazole monohydrate (HOBT) (143 mg; 1.06
mmol) in dimethylformamide (DMF) (lOml) were added 1-(3-
dimethylaminopropyl)-3-ethylcarbodimimide (EDC)
hydrochloride (270 mg; 1.42 mmol), triethylamine (148 ul;
1.06 mmol) and 4-dimethylaminopyridine (DMAP) (5 mg; 0.04
mmol). The reaction mixture was stirred at room
temperature for 15 hours, and the solvent was concentrated
under reduced pressure. The residue was dissolved in
chloroform, and the mixture was washed, dried, and the
solvent was evaporated under reduced pressure. The residue
was purified by silica gel column chromatography (solvent;
chloroform : methanol = 50:1 to 10:1) to give the title
compound (228 mg, yield: 75 $)asa pale yellow solid.
IR (NujolTM); 3290, 1710, 1655 cm 1
ESI-MS; 854 (M+H)
( 2 ) Preparation of (1S,9S)-1-(glycyl-glycyl-L-phenyl-
alanyl-glycylamino)-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-
4-methyl-1H, 12H-benzo [de] pyrano [ 3 ' , 4 ' : 6, 7] indolizino [ 1, 2-
b]quinoline-10,13(9H,15H)-dione:
Gly-Gly-Phe-Gly
.,\NH O
Me / I N O - HCI
F \ N ~ O.
HsC2 OH
CA 02457056 2004-02-10
To a solution of (1S,9S)-1-(t-butoxycarbonyl-glycyl-
glycyl-L-phenylalanyl-glycylamino)-9-ethyl-5-fluoro-2,3-
dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano-
[3' , 4' : 6, 7] indolizino [1, 2-b] quinoline-10, 13 (9H, 15H) -dione
5 (220 mg; 0.258 mmol) in dioxane (4 ml) was added 4N
hydrogen chloride solution in dioxane (6 ml) under stirring
in an ice bath. The mixture was stirred at room
temperature for 16 hours. Diethyl ether (30 ml) was added
to the reaction mixture, and the mixture was stirred at
10 room temperature for one hour. The precipitates were
collected by filtration, and dried to give the title
compound (176 mg, yield; 86 %) as a yellow powder.
IR (Nujol) ; 3250, 1745, 1660, 1605, 1535 cm'
ESI-MS; 754 (M+H)
15 (3) Preparation of dextran polyalcohol (PA-Dextran):
Acetic buffer (0.1 M, pH 5.5, 1000 ml) was put into a
three-neck round bottom flask (capacity; 3 liters).
Dextran T-500 (10.0 g, manufactured by Amersham Pharmacia
Biotech AB) was added in small portions to the buffer over
20 a period of 30 minutes at room temperature. The mixture
was stirred for about 30 minutes until the solution became
clear, and then, the mixture was cooled at 5 C (inner
temperature) in a bath.
Separately, to a flask (capacity; 1 liter) were added
25 sodium periodate (33.0 g) and water (1000 ml), and the
CA 02457056 2004-02-10
31
mixture was s.tirred at room temperature, and then -cooled at
C.
To the above dextran solution was added with stirring
the above sodium periodate solution at 5 C, and the mixture
5 was kept at 5 C for 5 days in a dark place. The excess
sodium periodate was removed by adding ethylene glycol (10
ml), and the mixture was further stirred at 5 C for 2 hours.
The reaction mixture was cooled to 3 C, and thereto was
added 8M aqueous sodium hydroxide solution during which the
reaction temperature was kept below 6 C (the pH value of
the reaction mixture became over pH 9). To the reaction
mixture was added sodium borohydride (14 g) in small
portions with stirring, and the mixture was stirred at 5 C
overnight. In order to remove the excess sodium
borohydride, -the pH value of the reaction mixture was
adjusted to below pH 5.5 by adding acetic acid thereto at 3
to 6 C, and the mixture was further stirred for 2 hours.
The pH value of the reaction mixture was adjusted to about
pH 7.8 with 8M aqueous sodium hydroxide solution. The
mixture was subjected to dialysis against water
(Spectora /Por 3 membrane, Molecule weight cutoff <3500),
and lyophilized to give dextran polyalcohol (8.34 g) as an
amorphous powder.
(4) Preparation of carboxymethyldextran polyalcohol (CM-
PA-Dextran):
CA 02457056 2004-02-10
WO 03/015826 PCT/JP02/08309
32
Water (155 ml) was put into a three-neck round bottom
flask (capacity; 500 ml), and thereto was added with
stirring dextran polyalcohol (5.18 g) at room temperature
over a period of 10 minutes. The mixture was stirred for
about 10 to 30 minutes until the mixture became clear, and
then sodium hydroxide (pellet, 97.0 %, 21*.8 g) was added to
the dextran polyalcohol solution in small portions under
stirring, during which the inner temperature was kept at 30
to 40 C in an ice bath. The reaction flask was put in a
bath, and the mixture was stirred at 30 C. Chloroacetic
acid (31.1 g) was added with stirring in small portions
into the reaction mixture at 30 to 40 C. After the
addition, the mixture was further stirred at 30 C in a bath
for 20 hours. The reaction mixture was cooled in an ice
bath, and the mixture was neutralized by adding thereto
acetic acid under stirring (i.e., the pH value was adjusted
to below pH 9).
Water (160 ml) was added to the mixture, and the
mixture was subjected to dialysis against water
(Spectora(D/Por 3 membrane, Molecule weight cutoff <3500),
and lyophilized to give carboxymethydextran polyalcohol
(6.53 g)as an amorphous powder.
(5) Preparation of carboxymethyldextran-polyalcohol-
(1S,9S)-1-(glycyl-glycyl-L-phenylalanyl-glycylamino)-9-
ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-
CA 02457056 2004-02-10
33
benzo [de]pyrano [3', 4' : 6, 7] indolizino [1, 2-b] quinoline-
10,13(9H,15H)-dione:
Water(40 ml) was put into a round bottom flask
(capacity; 100 ml), and thereto was added
carboxymethyldextran polyalcohol (1.Og) at room temperature
with stirring over a period of 5 minutes. The mixture was
stirred about 30 minutes until the mixture became clear. A
solution of (iS,9S)-1-(glycyl-glycyl-L-phenylalanyl-
glycylamino)-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-
methyl-1H, 12H-benzo [de]pyrano [3', 4' : 6, 7] indolizino [1, 2-
b]quinoline-10,13(9H,15H)-dione in dimethylformamide (100
mg/10 ml) was added with stirring to the mixture, and
further added thereto dimethylformamide (15 ml), and the
mixture was stirred for 10 minutes. To the mixture was
added dropwise with stirring a solution of 2-ethoxy-l-
ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) in dimethyl-
formamide (1.0 g/10 ml) at room temperature, and the
mixture was further stirred for 18 hours. The reaction
mixture was subjected to dialysis against water
(Spectora(D/Por 3 membrane, Molecule weight cutoff <3500),
and further purified by cation exchange column (BioRad AGa
MP-50 column, Na-type, 30 ml). The main fraction was
subjected to dialysis (Spectora /Por 3 membrane, Molecule
weight cutoff <3500), and lyophilized to give a crude
product, which was pulverized with acetone, collected by
CA 02457056 2004-02-10
34
filtration, and dried to give the desired product (904 mg)
as a pale yellow powder.
INDUSTRIAL APPLICABILITY
The pharmaceutical composition of the present
invention may accumulate highly at a site such as lymph
nodes or the liver to which cancers may spread, and
suppressively act on the growth of tumor cells without
affecting the normal cells, and hence, the
pharmaceutical composition of the present invention is
useful in the inhibition of metastasis, particularly in the
inhibition of lymph node metastasis or liver metastasis, or
prevention of reoccurrence of a malignant tumor.
In addition, the present pharmaceutical composition
may exhibit its effects not only before the onset of
metastasis but also after the onset of metastasis.
Therefore, the present pharmaceutical composition is also
useful in the inhibition of metastasis or prevention of
reoccurrence of a malignant tumor after topical therapy
(e.g., surgery, radiation therapy, thermotherapy,-
cryotherapy, laser burning therapy, etc.).
CA 02457056 2004-07-28
SEQUENCE LISTING
<110> Tanabe Seiyaku Co., Ltd.
<120> Pharmaceutical compositions comprising polysaccharide conjugates
for inhibiting the metastasis or preventing the recurrence of
malignant tumor.
<130> 57328-NP
<140> CA2457056
<141> 2002-08-16
<150> PCT/JP02/08309
<151> 2002-08-16
<150> JP 2001-249717
<151> 2001-08-21
<160> 3
<170> PatentIn version 3.2
<210> 1
<211> 4
<212> PRT
<213> Artificial sequence
<220>
<223> Peptide moiety or linker
<220>
<221> miscfeature
<222> (1) ._(1)
<223> Xaa is glycine or a modified amino acid to provide a compound
in accordance with the invention
<220>
<221> miscfeature
<222> (4)._(4)
<223> Xaa is glycine or a modified amino acid to provide a compound
in accordance with the invention
<400> 1
Xaa Gly Gly Xaa
1
<210> 2
<211> 5
<212> PRT
<213> Artificial sequence
<220>
<223> Peptide moiety or linker
<220>
<221> miscfeature
<222> (1)._(1)
<223> Xaa is glycine or a modified amino acid to provide a compound
in accordance with the invention
CA 02457056 2004-07-28
36
<220>
<221> misc_feature
<222> (5)..(5)
<223> Xaa is glycine or a modified amino acid to provide a compound
in accordance with the invention
<400> 2
Xaa Gly Gly Gly Xaa
1 5
<210> 3
<211> 4
<212> PRT
<213> Artificial sequence
<220>
<223> Preparation 4 of carboxymethyldextran-polyalcohol-(1S,9S)-1-
(glycyl-glycyl-L-phenylalanyl-glycylam
ino)-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-
benzo[de]pyrano-[3',4':6,7]indolizino[1,2-b]
quinoline-10,13(9H,15H)-dione and intermediates thereof
<220>
<221> miscfeature
<222> (1) .. (1)
<223> Xaa is a moiety to provide any one of the compounds
carboxymethyldextran-polyalcohol-(1S,9S)
-1-(glycyl-glycyl-L-phenylalanyl-glycylamino)-9-ethyl-5-fluoro-
2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo
[de]pyrano-[3',4':6,7]indolizino[1,2-b]quinoline-10,13(9H,15H)-
dione; (1S,9S)-1-(t-butoxycarbonyl-glycyl-
glycyl-L-phenylalanyl-glycylamino)-9-ethyl-5-fluoro-2,3-dihydro-
9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano-
[3',4':6,7]indolizino[1,2-b]quinoline-10,13(9H,15H)-dione; t-
butoxycarbonyl-glycyl-glycyl-L-phenylalanyl-
glycine; and (1S,9S)-1-(glycyl-glycyl-L-phenylalanyl-glycylamino)-
9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-
methyl-1H,12H-benzo[de]pyrano-[3',4':6,7]indolizino[1,2-b]
quinoline-10,13(9H,15H)-dione;
<220>
<221> miscfeature
<222> (4)..(4)
<223> Xaa is glycine or a moiety to provide any one of the compounds
carboxymethyldextran-polyalcohol-(1S,9S)
-1-(glycyl-glycyl-L-phenylalanyl-glycylamino)-9-ethyl-5-fluoro-
2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo
[de]pyrano-[3',4':6,7]indolizino[1,2-b]quinoline-10,13(9H,15H)-
dione; (1S,9S)-1-(t-butoxycarbonyl-glycyl-
glycyl-L-phenylalanyl-glycylamino)-9-ethyl-5-fluoro-2,3-dihydro-
9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano-
[3',4':6,7]indolizino[1,2-b]quinoline-10,13(9H,15H)-dione; t-
butoxycarbonyl-glycyl-glycyl-L-phenylalanyl-
glycine; and (1S,9S)-1-(glycyl-glycyl-L-phenylalanyl-glycylamino)-
9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-
methyl-1H,12H-benzo[de]pyrano-[3',4':6,7]indolizino[1,2-b]
quinoline-10,13(9H,15H)-dione;
CA 02457056 2004-07-28
37
<400> 3
Xaa Gly Phe Xaa
1
