U.S. patent application number 11/281773 was filed with the patent office on 2006-04-13 for cell division inhibitor and a production method thereof.
Invention is credited to Kazumi Akazawa, Kaneo Kanoh, Hiroshi Kanzaki, Teruhiko Nitoda, Satohiro Yanagisawa.
Application Number | 20060079534 11/281773 |
Document ID | / |
Family ID | 18537534 |
Filed Date | 2006-04-13 |
United States Patent
Application |
20060079534 |
Kind Code |
A1 |
Kanzaki; Hiroshi ; et
al. |
April 13, 2006 |
Cell division inhibitor and a production method thereof
Abstract
The present invention relates to a cell division inhibitor
comprising various dehydrodiketopiperazines such as
dehydrophenylahistin, or analogs thereof as an active ingredient,
and a dehydrogenase and a method for producing the same
inhibitor.
Inventors: |
Kanzaki; Hiroshi;
(Okayama-shi, JP) ; Kanoh; Kaneo; (Kamaishi-shi,
JP) ; Yanagisawa; Satohiro; (Kobe-shi, JP) ;
Nitoda; Teruhiko; (Okayama-shi, JP) ; Akazawa;
Kazumi; (Kurashiki-shi, JP) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
18537534 |
Appl. No.: |
11/281773 |
Filed: |
November 17, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10181786 |
Dec 2, 2002 |
6972289 |
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PCT/JP00/06807 |
Sep 29, 2000 |
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11281773 |
Nov 17, 2005 |
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Current U.S.
Class: |
514/254.05 ;
544/370 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 31/496 20130101; A61P 43/00 20180101; A61P 37/06 20180101;
A61P 29/00 20180101 |
Class at
Publication: |
514/254.05 ;
544/370 |
International
Class: |
A61K 31/496 20060101
A61K031/496; C07D 403/02 20060101 C07D403/02 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 18, 2000 |
JP |
2000-9370 |
Claims
1-14. (canceled)
15. A compound of formula (I) or pharmaceutically acceptable salt
thereof: ##STR8## wherein: each of X.sub.1 and X.sub.2 is
independently oxygen or sulfur; Y.sub.3 is oxygen, sulfur,
--NR.sub.3--, or --CR.sub.31R.sub.32--; Y.sub.4 is oxygen, sulfur,
--NR.sub.4--, or --CR.sub.41R.sub.42--; R.sub.10 is an aryl or
aralkyl and is optionally substituted with other substituent(s) and
optionally comprises one or more heteroatoms; R.sub.20 is an aryl
or aralkyl and is optionally substituted with other substituent(s)
and optionally comprises one or more heteroatoms, with the proviso
that R.sub.20 does not include a phenyl group; each of R.sub.3 and
R.sub.4 is independently selected from the group consisting of
hydrogen, halogen, C.sub.1-25 alkyl, C.sub.2-25 alkenyl, C.sub.2-25
alkynyl, C.sub.1-25 alkoxy, aralkyl, hydroxyl, amino, nitro, and
aryl, each of which is optionally substituted with other
substituent(s), wherein any part of a carbon chain in R.sub.3 and
R.sub.4 may be branched or cyclized, and may comprise one or more
heteroatoms; each of R.sub.31, R.sub.32, R.sub.41, and R.sub.42 is
independently selected from the group consisting of hydrogen,
halogen, C.sub.1-25 alkyl, C.sub.2-25 alkenyl, C.sub.2-25 alkynyl,
C.sub.1-25 alkoxy, aralkyl, hydroxyl, amino, nitro, and aryl, each
of which is optionally substituted with other substituent(s),
wherein any part of a carbon chain in R.sub.31, R.sub.32, R.sub.41,
and R.sub.42 may be branched or cyclized, and may comprise one or
more heteroatoms; R.sub.10 and any of R.sub.3, R.sub.31, or
R.sub.32 may together from a ring; R.sub.20 and any of R.sub.4,
R.sub.41, or R.sub.42 may together from a ring; each bond depicted
by a solid line and dashed line represents a carbon-carbon single
bond or a carbon-carbon double bond with E or Z configurations; and
at least one of said groups may have a protecting group capable of
decomposing in vivo; with the proviso that the compound of formula
I does not include the compound having the following structure:
##STR9##
16. The compound of claim 15, wherein each bond depicted by a solid
line and dashed line represents a carbon-carbon double bond.
17. The compound of claim 16, wherein each of X.sub.1 and X.sub.2
is oxygen, Y.sub.3 is --NR.sub.3--, and Y.sub.4 is
--NR.sub.4--.
18. The compound of claim 17, wherein each of Y.sub.3 and Y.sub.4
is --NH--.
19. The compound of claim 15, wherein at least one of R.sub.10 and
R.sub.20 is substituted with 1,1-dimethyl-2-propenyl.
20. The compound of claim 15, wherein at least one of R.sub.10 and
R.sub.20 has the following structure: ##STR10## and is optionally
substituted with other substituent(s).
21. The compound of claim 15, wherein: R.sub.10 is a phenyl or
phenylalkyl and is optionally substituted with other
substituent(s); and R.sub.20 is an imidazolyl or imidazolylalkyl
and is optionally substituted with other substituent(s).
22. The compound of claim 15 selected from the group consisting of:
3-(imidazole-4-ylmethylene)-6-(phenylmethylene)piperazine-2,5-dione;
3-[(5-methylimidazole-4-yl)methylene]-6-(phenylmethylene)piperazine-2,5-d-
ione;
3-[(5-ethylimidazole-4-yl)methylene]-6-(phenylmethylene)piperazine--
2,5-dione;
3-[(5-butylimidazole-4-yl)methylene]-6-(phenylmethylene)piperazine-2,5-di-
one;
3-[(5-pentylimidazole-4-yl)methylene]-6-(phenylmethylene)piperazine--
2,5-dione; and 3-{[5-(1,
1-dimethyl-2-propenyl)imidazole-4-yl]methylene}-6-(phenylmethylene)
piperazine-2,5-dione.
23. A method of inhibiting cell division in a subject, comprising
administering to the subject a compound of claim 15.
24. The method of claim 23, wherein the compound is selected from
the group consisting of:
3-(imidazole-4-ylmethylene)-6-(phenylmethylene)piperazine-2,5-dione;
3-[(5-methylimidazole-4-yl)methylene]-6-(phenylmethylene)piperazine-2,5-d-
ione;
3-[(5-ethylimidazole-4-yl)methylene]-6-(phenylmethylene)piperazine--
2,5-dione;
3-[(5-butylimidazole-4-yl)methylene]-6-(phenylmethylene)piperazine-2,5-di-
one; and
3-[(5-pentylimidazole-4-yl)methylene]-6-(phenylmethylene)piperaz-
ine-2,5-dione.
25. The method of claim 23, wherein the compound is
3-{[5-(1,1-dimethyl-2-propenyl)imidazole-4-yl]methylene}-6-(phenylmethyle-
ne)piperazine-2,5-dione.
26. A method of treating a subject having a tumor, comprising
administering to the subject a compound of claim 15.
27. The method of claim 26, wherein the compound is selected from
the group consisting of:
3-(imidazole-4-ylmethylene)-6-(phenylmethylene)piperazine-2,5-dione;
3-[(5-methylimidazole-4-yl)methylene]-6-(phenylmethylene)piperazine-2,5-d-
ione;
3-[(5-ethylimidazole-4-yl)methylene]-6-(phenylmethylene)piperazine--
2,5-dione;
3-[(5-butylimidazole-4-yl)methylene]-6-(phenylmethylene)piperazine-2,5-di-
one; and
3-[(5-pentylimidazole-4-yl)methylene]-6-(phenylmethylene)piperaz-
ine-2,5-dione.
28. The method of claim 26, wherein the compound is
3-{[5-(1,1-dimethyl-2-propenyl)imidazole-4-yl]methylene}-6-(phenylmethyle-
ne)piperazine-2,5-dione.
Description
TECHNICAL FIELD
[0001] The present invention relates to a cell division inhibitor
(a cell cycle inhibitor) and an antitumor agent, and a method of
producing them using enzymes.
BACKGROUND
[0002] The growth and differentiation of cells constituting a human
body are strictly controlled in order to maintain homeostasis.
Cells divide or proliferate by repeating a cell cycle consisting of
a certain process comprising M period, G1 period, S period and G2
period. A defect in the control mechanism of this cell cycle
results in the development of cancer or immune disease.
[0003] Lately, the control mechanism of cell cycle is clarifying at
a molecular level, and it is known that a substance controlling a
cell cycle possibly can be used as an antitumor agent or an
immunosuppressive agent. In recent years, as an antitumor agent or
a lead compound thereof, the spotlight has centered on a substance,
such as pacritaxel, vincristine or vinblastine, inhibiting the
function of tubulin which is one of cytoskeleton proteins playing a
major role in precisely distributing a replicated gene into a
daughter cell in a cell division stage.
[0004] Fukushima et al. have found that albonoursin has an
antitumor activity and an antibacterial activity (Fukushima et al.,
J. Antibiotics, Vol. 26, pp. 175, 1973), while Kobayashi et al.
have found that albonoursin acts to inhibit the pronuclear fusion
between a female nucleus and a male nucleus (Kobayashi et al., A
Summary of the Symposium on the Chemistry of Natural Products, P51,
1989). Furthermore, Kanzaki et al. have found that
tetradehydrocyclo (Phe-Phe) exhibits sea urchin embryo division
inhibitory activity (A Summary of the Symposium of the Society for
Actinomycetes Japan, P42, 1999).
[0005] Kanoh et al. have found that, filamentous bacteria,
Aspergillus ustus NSC-F037 and Aspergillus ustus NSC-F038, which
were isolated from the soil in Kanagawa prefecture, produce a novel
antitumor substance phenylahistin, and have determined the
structure of this substance. Phenylahistin molecules have a chiral
carbon atom, and as a result of a thorough examination, Kanoh et
al. have further discovered that phenylahistin produced by the
above bacteria is a mixture of (-)-phenylahistin and
(+)-phenylahistin, and that the antitumor acitivity of
(-)-phenylahistin is approx. 30-100 times stronger than that of
(+)-phenylahistin (Japanese Patent Application Laying-Open (kokai)
No. 10-130266, Kanoh et al., Bioorganic & Medicinal Chemistry
Letters, Vol. 7, No. 22, pp. 2847-2852, 1997; Kanoh et al.,
Bioscience Biotechnology Biochemistry, Vol. 63, No. 6, pp.
1130-1133, 1999). Further, they have found that (-)-phenylahistin
inhibits the polymerization of tubulin (Kanoh et al., The Journal
of Antibiotics, Vol. 52, No. 2, pp. 134-141, 1999). Furthermore,
Kanoh et al. have examined the antitumor effect of
(-)-phenylahistin, using a cancer cell transplanted model animal,
and have shown that (-)-phenylahistin has a certain degree of
antitumor activity (Kanoh et al., Bioscience Biotechnology
Biochemistry, Vol. 63, No. 6, pp. 1130-1133, 1999). From a clinical
position, however, an agent having a stronger antitumor activity
than (-)-phenylahistin is desirable.
DISCLOSURE OF THE INVENTION
[0006] The object of the present invention is to provide a cell
division inhibitor having a stronger cell cycle inhibitory
activity, particularly antitumor activity, and a method of
producing the inhibitor using enzymes.
[0007] As a result of thorough analysis by the present inventors to
achieve the above object, it has been found that various
dehydrodiketopiperazines such as dehydrophenylahistin or affinities
thereof have a stronger cell cycle inhibitory activity than
(-)-phenylahistin, and have completed the present invention.
[0008] That is to say, the present invention comprises each of the
following inventions. [0009] (1) A cell division inhibitor
comprising, as an active ingredient, a compound of formula (I) or
pharmaceutically acceptable salt thereof: ##STR1## wherein [0010]
each of X.sub.1 and X.sub.2 is independently oxygen or sulfur;
[0011] Y.sub.3 is oxygen, sulfur, --NR.sub.3-- or
--CR.sub.31R.sub.32--; [0012] Y.sub.4 is oxygen, sulfur,
--NR.sub.4-- or --CR.sub.41R.sub.42--; [0013] R.sub.10 is halogen,
C.sub.1-25 alkyl, C.sub.2-25 alkenyl, C.sub.2-25 alkynyl,
C.sub.1-25 alkoxy, aralkyl, hydroxyl, amino, nitro or aryl, which
may be substituted with other substituent(s), and a part of the
carbon chain of R.sub.10 may be branched or cyclized, or may
comprise a heteroatom; [0014] R.sub.20 is halogen, C.sub.1-2 alkyl,
C.sub.2-25 alkenyl, C.sub.2-25 alkynyl, C.sub.1-25 alkoxy, aralkyl,
hydroxyl, amino, nitro or aryl, which may be substituted with other
substituent(s), and a part of the carbon chain of R.sub.20 may be
branched or cyclized, or may comprise a heteroatom; each of R.sub.3
and R.sub.4 is independently hydrogen, halogen, C.sub.1-25 alkyl,
C.sub.2-25 alkenyl, C.sub.2-25 alkynyl, C.sub.1-25 alkoxy, aralkyl,
hydroxyl, amino, nitro or aryl, which may be substituted with other
substituent(s), and a part of the carbon chain may be branched or
cyclized, or may comprise a heteroatom; [0015] each of R.sub.31,
R.sub.32, R.sub.41 and R.sub.42 is independently hydrogen, halogen,
C.sub.1-25 alkyl, C.sub.2-25 alkenyl, C.sub.2-25 alkynyl,
C.sub.1-25 alkoxy, aralkyl, hydroxyl, amino, nitro or aryl, which
may be substituted with other substituent(s), and a part of the
carbon chain may be branched or cyclized, or may comprise a
heteroatom; [0016] R.sub.10 and any of R.sub.3, R.sub.31 and
R.sub.32 may form a ring; [0017] R.sub.20 and any of R.sub.4,
R.sub.41 and R.sub.42 may form a ring; [0018] each of (B1) and (B2)
independently represents a carbon-carbon single bond or a
carbon-carbon double bond, wherein at least one represents a
carbon-carbon double bond with E or Z configuration; [0019] at
least one of the above groups may have a protecting group capable
of decomposing in vivo, except in the case where each of X.sub.1
and X.sub.2 is oxygen, each of Y.sub.3 and Y.sub.4 is --NH--,
R.sub.10 is benzyl, each of (B1) and (B2) is a carbon-carbon double
bond, and R.sub.20 is isobutyl or benzyl, and in the case where
each of X.sub.1 and X.sub.2 is oxygen, each of Y.sub.3 and Y.sub.4
is --NH--, R.sub.10 is benzyl, (B1) is a carbon-carbon single bond,
(B2) is a carbon-carbon Z double bond, and R.sub.20 is a group
shown in the following formula (a): ##STR2## wherein * represents a
bonding position.
[0020] (2) The cell division inhibitor according to item 1 above
wherein, in the formula (I), each of (B1) and (B2) is a
carbon-carbon double bond.
[0021] (3) The cell division inhibitor according to item 1 or 2
above wherein, in the formula (I), each of X.sub.1 and X.sub.2 is
oxygen, Y.sub.3 is --NR.sub.3--, and Y.sub.4 is --NR.sub.4--.
[0022] (4) The cell division inhibitor according to item 3 above
wherein, in the formula (I), each of Y.sub.3 and Y.sub.4 is
--NH--.
[0023] (5) A cell division inhibitor comprising, as an active
ingredient, a compound of formula (II) or an E form thereof, or
pharmaceutically acceptable salt thereof: ##STR3## wherein [0024]
R.sub.1 is hydrogen, halogen, C.sub.1-25 alkyl, C.sub.2-25 alkenyl,
C.sub.2-25 alkynyl, C.sub.1-25 alkoxy, aralkyl, hydroxyl, amino,
nitro or aryl, which may be substituted with other substituent(s),
and a part of the carbon chain of R.sub.1 may be branched or
cyclized, or may comprise a heteroatom, and further R.sub.1 may be
one atom or group, or at most 5 identical or different atoms or
groups, and the atoms or groups may mutually form a ring; [0025]
R.sub.2 is hydrogen, halogen, C.sub.1-25, alkyl, C.sub.2-25
alkenyl, C.sub.2-25 alkynyl, C.sub.1-25, alkoxy, aralkyl, hydroxyl,
amino, nitro or aryl, which may be substituted with other
substituent(s), and a part of the carbon chain of R.sub.2 may be
branched or cyclized, or may comprise a heteroatom; [0026] each of
R.sub.3 and R.sub.4 is independently hydrogen, halogen, C.sub.1-25
alkyl, C.sub.2-25 alkenyl, C.sub.2-25 alkynyl, C.sub.1-25 alkoxy,
aralkyl, hydroxyl, amino, nitro or aryl, which may be substituted
with other substituent(s), and a part of the carbon chain may be
branched or cyclized, or may comprise a heteroatom; [0027] R.sub.5
is hydrogen, halogen, C.sub.1-25 alkyl, C.sub.2-25 alkenyl,
C.sub.2-25 alkynyl, C.sub.1-25 alkoxy, aralkyl, hydroxyl, amino,
nitro or aryl, which may be substituted with other substituent(s),
and a part of the carbon chain of R.sub.5 may be branched or
cyclized, or may comprise a heteroatom; [0028] R.sub.6 is hydrogen,
halogen, C.sub.1-25 alkyl, C.sub.2-25 alkenyl, C.sub.2-25 alkynyl,
C.sub.1-25 alkoxy, aralkyl, hydroxyl, amino, nitro or aryl, which
may be substituted with other substituent(s), and a part of the
carbon chain of R.sub.6 may be branched or cyclized, or may
comprise a heteroatom; [0029] each of R.sub.7 and R.sub.8 is
independently hydrogen, halogen, C.sub.1-25 alkyl, C.sub.2-25
alkenyl, C.sub.2-25 alkynyl, C.sub.1-25 alkoxy, aralkyl, hydroxyl,
amino, nitro or aryl, which may be substituted with other
substituent(s), and a part of the carbon chain may be branched or
cyclized, or may comprise a heteroatom; [0030] R.sub.2 and R.sub.3
may form a ring; [0031] R.sub.4 and any of R.sub.5, R.sub.6,
R.sub.7 and R.sub.8 may form a ring; [0032] (B2) represents a
carbon-carbon single bond or a carbon-carbon double bond; [0033] at
least one of the above groups may have a protecting group capable
of decomposing in vivo.
[0034] (6) The cell division inhibitor according to item 5 above
wherein, in the formula (II), (B2) is a carbon-carbon double
bond.
[0035] (7) The cell division inhibitor according to item 6 above
wherein, in the formula (II), at least one of R.sub.7 and R.sub.8
is 1,1-dimethyl-2-propenyl.
[0036] (8) The cell division inhibitor according to any one of
items 1-7 above wherein it is an antitumor agent.
[0037] (9) A dehydrogenase which has an activity to convert a
compound represented by the above formula (I) wherein at least one
of (B1) and (B2) is a carbon-carbon single bond, or by the above
formula (II) wherein (B2) is a carbon-carbon single bond into a
compound wherein the carbon-carbon single bond(s) is replaced with
a carbon-carbon double bond(s).
[0038] (10) The dehydrogenase according to item 9 above whose
molecular weight is 700-800 kDa.
[0039] (11) The dehydrogenase according to item 9 or 10 above which
is produced by Streptomyces albulus.
[0040] (12) A method of producing the cell division inhibitor
according to any one of items 1-8 above, which comprises using, as
a substrate, a compound represented by the above formula (I)
wherein at least one of (B1) and (B2) is a carbon-carbon single
bond, or a compound represented by the above formula (II) wherein
(B2) is a carbon-carbon single bond, and converting the
carbon-carbon single bond to a carbon-carbon double bond by use of
a cell, cell-free extract or enzyme solution containing the
dehydrogenase according to any one of items 9-11 above.
[0041] (13) The method according to item 12 above wherein the
dehydrogenase of item 11 above is used.
[0042] (14) A compound of formula (II) or an E form thereof, or
pharmaceutically acceptable salt thereof: ##STR4## wherein [0043]
R.sub.1 is hydrogen, halogen, C.sub.1-25 alkyl, C.sub.2-25 alkenyl,
C.sub.2-25 alkynyl, C.sub.1-25 alkoxy, aralkyl, hydroxyl, amino,
nitro or aryl, which may be substituted with other substituent(s),
and a part of the carbon chain of R.sub.1 may be branched or
cyclized, or may comprise a heteroatom, and further R.sub.1 may be
one atom or group, or at most 5 identical or different atoms or
groups, and the atoms or groups may mutually form a ring; [0044]
R.sub.2 is hydrogen, halogen, C.sub.1-25 alkyl, C.sub.2-25 alkenyl,
C.sub.2-25 alkynyl, C.sub.2-25 alkoxy, aralkyl, hydroxyl, amino,
nitro or aryl, which may be substituted with other substituent(s),
and a part of the carbon chain of R.sub.2 may be branched or
cyclized, or may comprise a heteroatom; [0045] each of R.sub.3 and
R.sub.4 is independently hydrogen, halogen, C.sub.1-25 alkyl,
C.sub.2-25 alkenyl, C.sub.2-25 alkynyl, C.sub.1-25 alkoxy, aralkyl,
hydroxyl, amino, nitro or aryl, which may be substituted with other
substituent(s), and a part of the carbon chain may be branched or
cyclized, or may comprise a heteroatom; [0046] R.sub.5 is hydrogen,
halogen, C.sub.1-25 alkyl, C.sub.2-25 alkenyl, C.sub.2-25 alkynyl,
C.sub.1-25 alkoxy, aralkyl, hydroxyl, amino, nitro or aryl, which
may be substituted with other substituent(s), and a part of the
carbon chain of R.sub.5 may be branched or cyclized, or may
comprise a heteroatom; [0047] R.sub.6 is hydrogen, halogen,
C.sub.1-25 alkyl, C.sub.2-25 alkenyl, C.sub.2-25 alkynyl,
C.sub.1-25 alkoxy, aralkyl, hydroxyl, amino, nitro or aryl, which
may be substituted with other substituent(s), and a part of the
carbon chain of R.sub.6 may be branched or cyclized, or may
comprise a heteroatom; [0048] each of R.sub.7 and R.sub.8 is
independently hydrogen, halogen, C.sub.1-25 alkyl, C.sub.2-25
alkenyl, C.sub.2-25 alkynyl, C.sub.1-25 alkoxy, aralkyl, hydroxyl,
amino, nitro or aryl, which may be substituted with other
substituent(s), and a part of the carbon chain may be branched or
cyclized, or may comprise a heteroatom; [0049] R.sub.2 and R.sub.3
may form a ring; [0050] R.sub.4 and any of R.sub.5, R.sub.6,
R.sub.7 and R.sub.8 may form a ring; [0051] (B2) represents a
carbon-carbon single bond or a carbon-carbon double bond; [0052] at
least one of the above groups may have a protecting group capable
of decomposing in vivo.
[0053] The details of the present invention are disclosed
below.
[0054] First of all, regarding various definitions which the
present invention comprises, appropriate examples and explanations
are provided below.
[0055] The term "halogen" appearing in the formulas (I) and (II)
means fluorine, chlorine, bromine or iodine, unless otherwise
specified.
[0056] C.sub.1-25 alkyl represented by R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.10, R.sub.20,
R.sub.31, R.sub.32, R.sub.41 or R.sub.42 is an alkyl group having 1
to 25 carbon atoms, which may be normal-chained, branched or
cyclized. Examples of C.sub.1-25 alkyl include methyl, ethyl,
propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl,
pentyl, isopentyl, cyclopentyl, hexyl, cyclohexyl, heptyl,
5-methylhexyl, cycloheptyl, octyl, 6-methylheptyl, nonyl,
7-methyloctyl, decyl and 8-methylnonyl, preferably C.sub.1-10
alkyl, and more preferably C.sub.1-6 alkyl. These alkyl groups may
be substituted with other substituent(s), and may comprise a
heteroatom such as halogen, oxygen, sulfur, nitrogen or the
like.
[0057] C.sub.2-25 alkenyl represented by R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.10, R.sub.20,
R.sub.31, R.sub.32, R.sub.41 or R.sub.42 is an alkenyl group having
2 to 25 carbon atoms, which may be normal-chained, branched or
cyclized. Examples of C.sub.2-25 alkenyl include vinyl, propenyl,
1,1-dimethyl-2-propenyl and 3-methyl-3-butenyl, preferably
C.sub.2-10 alkenyl, and more preferably C.sub.2-6 alkenyl. These
alkenyl groups may be substituted with other substituent(s), and
may comprise a heteroatom such as halogen, oxygen, sulfur, nitrogen
or the like.
[0058] C.sub.2-25 alkynyl represented by R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.10, R.sub.20,
R.sub.31, R.sub.32, R.sub.41 or R.sub.42 is an alkynyl group having
2 to 25 carbon atoms, which may be normal-chained, branched or
cyclized. Examples of C.sub.2-25 alkynyl include ethynyl, propynyl
and butynyl, preferably C.sub.2-10 alkynyl, and more preferably
C.sub.2-6 alkynyl. These alkenyl groups may be substituted with
other substituent(s), and may comprise a heteroatom such as
halogen, oxygen, sulfur, nitrogen or the like.
[0059] C.sub.1-25 alkoxy represented by R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.10, R.sub.20,
R.sub.31, R.sub.32, R.sub.41 or R.sub.42 is an alkoxy group having
1 to 25 carbon atoms, which may be normal-chained, branched or
cyclized. Examples of C.sub.1-25 alkoxy include methoxy, ethoxy,
propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentyloxy,
isopentyloxy, cyclopentyloxy, hexyloxy, cyclohexyloxy, heptyloxy,
5-methylhexyloxy, cycloheptyloxy, octyl, 6-methylheptyloxy,
nonyloxy, 7-methyloctyloxy, decyloxy and 8-methylnonyloxy,
preferably C.sub.1-10 alkoxy, and more preferably C.sub.1-6 alkoxy.
These alkoxy groups may be substituted with other substituent(s),
and may comprise a heteroatom such as halogen, oxygen, sulfur,
nitrogen or the like.
[0060] Aryl represented by R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.10, R.sub.20, R.sub.31,
R.sub.32, R.sub.41 or R.sub.42 is a monocyclic or polycyclic
aromatic hydrocarbon group, and examples include phenyl, naphthyl
and anthranyl, but preferably phenyl. These aryl groups may be
substituted with other substituent(s) such as C.sub.1-6 alkyl
(preferably methyl, ethyl and propyl), C.sub.1-6 alkoxy, halogen,
nitro, amino, carboxyl, hydroxy-C.sub.1-6 alkyl, hydroxyl or
protected hydroxyl, and may comprise a heteroatom such as oxygen,
sulfur, nitrogen or the like as a ring forming member.
[0061] Aralkyl represented by R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.10, R.sub.20, R.sub.31,
R.sub.32, R.sub.41 or R.sub.42 is C.sub.1-6 alkyl substituted with
the above aryl, and examples include benzyl, phenethyl,
naphthylmethyl and anthranylmethyl, but preferably benzyl. These
aralkyl groups may be substituted with other substituent(s) such as
C.sub.1-6 alkyl (preferably methyl, ethyl and propyl), C.sub.1-6
alkoxy, halogen, nitro, amino, carboxyl, hydroxy-C.sub.1-6 alkyl,
hydroxyl or protected hydroxyl, and may comprise a heteroatom such
as oxygen, sulfur, nitrogen or the like as a ring forming
member.
[0062] The examples of substituents in substituted amino
represented by R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5,
R.sub.6, R.sub.7, R.sub.8, R.sub.10, R.sub.20, R.sub.31, R.sub.32,
R.sub.41 or R.sub.42 include C.sub.1-6 alkyl, C.sub.1-6 alkoxy,
halogen, carboxyl, hydroxy-C.sub.1-6 alkyl, hydroxyl or protected
hydroxyl.
[0063] In the above formula (I), R.sub.10 and any of R.sub.3,
R.sub.3, and R.sub.32 may form a ring, and R.sub.20 and any of
R.sub.4, R.sub.41 and R.sub.42 may form a ring. In the above
formula (II), R.sub.2 and R.sub.3 may form a ring, and R.sub.4 and
any of R.sub.5, R.sub.6, R.sub.7 and R.sub.8 may form a ring.
[0064] As C.sub.2-25 alkenyl represented by R.sub.7 or R.sub.8, an
alkenyl group corresponding to an isoprene unit consisting of 5
carbon atoms, that is, 1,1-dimethyl-2-propenyl or
3-methyl-3-butenyl, and an alkenyl group consisting of two or more
isoprene units, preferably at most 3 isoprene units (up to 15
carbon atoms) are desirable.
[0065] Substituents appearing in the above formulas (I) and (II)
may have a protecting group capable of decomposing in vivo. Among
these protecting groups, as a protecting group for an amino group
for example, there may be used a protecting group having the
binding form such as acid amide, carbamate and the like which are
described in Drug Development vol. 13, "Drug Delivery Systems"
edited by Hitoshi SEZAKI, Hirokawa Shoten (July 1989), page 116,
Table 2. 29, but an acyl such as acetyl derived from fatty acid is
preferable.
[0066] The double bond of a compound shown in the above formulas
(I) or (II) may be either in Z configuration or in E configuration,
but preferably in Z configuration.
[0067] In the case where (B1) and/or (B2) is a carbon-carbon double
bond, the above substituent binding to the above carbon-carbon
double bond becomes a corresponding divalent group. For example,
methyl becomes methylene, and benzyl becomes phenylmethylene
(benzylidene).
[0068] Among possible compounds represented by the above formula
(I), a compound wherein each of X.sub.1 and X.sub.2 is oxygen, each
of Y.sub.3 and Y.sub.4 is --NH--, R.sub.10 is benzyl, each of (B1)
and (B2) is a carbon-carbon double bond, and R.sub.20 is isobutyl
(the common name: albonoursin, the compound name:
3-(Z)-benzylidene-6-(Z)isobutylidene-2,5-piperazine dione) refers
to the known antitumor agent described in Fukushima et al., J.
Antibiotics, Vol. 26, pp. 175, 1973) and the known pronuclear
fusion inhibitory agent described in A Summary of the Symposium on
the Chemistry of Natural Products, P51, 1989, and these two agents
are excluded from the cell division inhibitor of the present
invention. In addition, a compound (tetradehydrocyclo (Phe-Phe))
wherein each of X.sub.1 and X.sub.2 is oxygen, each of Y.sub.3 and
Y.sub.4 is --NH--, each of R.sub.10 and R.sub.20 is benzyl, and
each of (B1) and (B2) is a carbon-carbon double bond refers to the
known sea urchin embryo division inhibitory agent described in A
Summary of the Symposium of the Society for Actinomycetes Japan,
P42, 1999, and this agent is excluded from the cell division
inhibitor of the present invention. Furthermore, a compound (the
common name: phenylahistin, the compound name:
3-{[5-(1,1-dimethyl-2-propenyl)imidazole-4-yl]methylene}-6-benzylpiperazi-
ne-2,5-dione) wherein each of X.sub.1 and X.sub.2 is oxygen, each
of Y.sub.3 and Y.sub.4 is --NH--, R.sub.10 is benzyl, (B1) is a
carbon-carbon single bond, (B2) is a carbon-carbon Z double bond,
and R.sub.20 is a group shown in the following formula (a):
##STR5## [0069] (wherein * represents a bonding position) [0070] is
the known cell division inhibitor disclosed in Japanese Patent
Application Laying-Open (kokai) No. 10-130266 and so on, and this
agent is excluded from the cell division inhibitor of the present
invention. Generally, a compound wherein, in the above formula (I),
each of X.sub.1 and X.sub.2 is independently oxygen or sulfur,
Y.sub.3 is --NR.sub.3--, Y.sub.4 is --NR.sub.4-- (herein R.sub.3
and R.sub.4 are defined as with stated above), R.sub.10 is
substituted or unsubstituted benzyl, (B1) is a carbon-carbon single
bond, (B2) is a carbon-carbon Z double bond, and R.sub.20 is
substituted or unsubstituted imidazole-4-ylmethylene is excluded
from those used for the cell division inhibitor of the present
invention.
[0071] Further, in the above formula (I), a compound wherein (B1)
is a carbon-carbon double bond and (B2) is a carbon-carbon single
bond or a carbon-carbon double bond is preferable, and a compound
wherein each of (B1) and (B2) is a carbon-carbon double bond is
more preferable.
[0072] Preferable examples of compounds shown in the above formulas
(I) and (II) include
3-(imidazole-4-ylmethylene)-6-(phenylmethylene)piperazine-2,5-dione;
3-[(5-methylimidazole-4-yl)methylene]-6-(phenylmethylene)piperazine-2,5-d-
ione;
3-[(5-ethylimidazole-4-yl)methylene]-6-(phenylmethylene)piperazine-2-
,5-dione;
3-[(5-butylimidazole-4-yl)methylene]-6-(phenylmethylene)piperazi-
ne-2,5-dione;
3-[(5-pentylimidazole-4-yl)methylene]-6-(phenylmethylene)piperazine-2,5-d-
ione;
3-[5-(1,1-dimethyl-2-propenyl)imidazole-4-yl]methylene)-6-(phenylmet-
hylene) piperazine-2,5-dione.
[0073] Pharmaceutically acceptable salt of a compound shown in the
above formula (I) or (II) is ordinary organic or inorganic atoxic
salt. In the case where the above compound is a basic substance,
the salts that are preferably used are hydrochloride, hydrobromide,
sulfate, nitrate, acetate, methanesulfonate and toluenesulfonate,
and in the case where the compound is an acidic substance, the salt
that is preferably used is a salt with inorganic base including
alkali metallic salt (e.g. sodium salt, potassium salt etc.) and
alkali-earth metallic salt (e.g. calcium salt, magnesium salt etc.)
The term "pharmaceutically acceptable" in the present specification
means that the salt is not only acceptable in medical agents,
veterinary agents, agricultural chemicals, antimicrobial agents,
insecticides etc., but also in a field comprising reagents used for
study purposes.
[0074] The cell division inhibitor of the present invention can be
used for the purpose of inhibiting the cell division, cell cycle
and pronuclear fusion between a female nucleus and a male nucleus
of a procaryote or an eucaryote. Specifically, the cell division
inhibitor of the present invention is useful as an antimicrobial
agent, agricultural chemical, veterinary agent, insecticide,
medical agent and reagent for study purposes. Furthermore, among
medical agents, it is particularly useful as an antitumor agent.
The cell division inhibitor of the present invention is effective
in a pathological condition wherein cell divisions are disorderly
repeated. It is particularly useful for cancers, and also effective
in a pathological condition appearing in a certain type of
autoimmune disease, chronic articular rheumatism etc., where a
certain type of cell continues to grow disorderly.
[0075] Furthermore, the antitumor agent of the present invention
can comprise other pharmaceutically effective ingredients, i.e.,
other antitumor agents as necessary as well as the above active
ingredients in order to treat various diseases. When the antitumor
agent takes the form of granule, fine granule, powder, tablet or
capsule, it is preferable that the antitumor agent comprises 5-80
weight % of the above active ingredients. When the antitumor agent
takes a liquid form, it is preferable that the antitumor agent
comprises 1-30 weight % of the above active ingredients. Further,
when the antitumor agent is used as an injection among parenteral
agents, it is preferable that the inhibitor comprises 1-10 weight %
of the above active ingredients.
[0076] For use in oral administration, the applied dose of the
above active ingredients is preferably 0.1 mg to 1 g per day per
adult. However, depending on the age, body weight, symptom etc. of
a patient, the dose can be changed as appropriate. The antitumor
agent of the present invention can be administered once per day,
but also it can be dividedly administered twice or three times at
regular time intervals. When it is used as an injection, the
applied dose of the above active ingredients is preferably 1 to
several hundreds of milligrams per administration per adult.
Moreover, it is possible to administer 1-3 times per day or once
every 2 or 3 days by injection, or to administer sustainably by
drip infusion or the like.
[0077] As a substrate of the dehydrogenase of the present invention
comprises a compound wherein, in the above formula (I), at least
one of (B1) and (B2) is a carbon-carbon single bond, or a compound
wherein, in the above formula (II), (B2) is a carbon-carbon single
bond can be used, but preferably a compound wherein, in the above
formula (I), each of X, and X.sub.2 is oxygen, Y.sub.3 is
--NR.sub.3--, and Y.sub.4 is --NR.sub.4-- (herein R.sub.3 and
R.sub.4 are defined as with stated above), more preferably a
compound wherein, in the above formula (I), each of X, and X.sub.2
is oxygen, and each of Y.sub.3 and Y.sub.4 is --NH-- is used, and
further more preferably a cyclic dipeptide wherein two amino acids
of L form condense to form a diketopiperazine ring or substituted
compounds thereof is used. Examples of the above condensing amino
acids preferably include cyclic (aromatic) amino acids such as
phenylalanine, histidine, tryptophan and tyrosine. Examples of
substituents in the substituted compounds of the above cyclic
dipeptide include halogen, C.sub.1-25 alkyl, C.sub.2-25 alkenyl,
C.sub.2-25 alkynyl, C.sub.1-25 alkoxy, aralkyl, hydroxyl, amino,
nitro and aryl. These substituents may be substituted with other
substituent(s), and a part of the carbon chain may be branched or
cyclized, may comprise a heteroatom, may mutually form a ring, and
may have a protecting group capable of decomposing in vivo. The
substituents include preferably C.sub.2-6 alkyl or C.sub.2-6
alkenyl, more preferably 1,1-dimethyl-2-propenyl.
[0078] The majority of compounds used as the substrate stated above
are the known compounds (Japanese Patent Application Laying-Open
(kokai) No. 10-130266; Kanoh et al., Bioorganic & Medicinal
Chemistry Letters, Vol. 7, No. 22, pp. 2847-2852, 1997; Kanoh et
al., Bioscience Biotechnology Biochemistry, Vol. 63, No. 6, pp.
1130-1133, 1999; Kanoh et al., Bioorganic & Medicinal
Chemistry, Vol. 7, pp. 1451-1457, 1999), and these compounds are
available. Other compounds can be produced by the same methods as
those described in Kopple et al., The Journal of Organic Chemistry,
Vol. 33, pp. 862-864, 1968 or Nitecki et al., The Journal of
Organic Chemistry, Vol. 33, pp. 864-866, 1968.
[0079] The dehydrogenase of the present invention includes
molecules having a variety of molecular weights, but one whose
molecular weight is 700-800 kDa is preferable.
[0080] The present invention can use, as a coenzyme of the
dehydrogenase, synthetic compounds such as dichlorophenolindophenol
(DCIP), phenazine methosulfate (PMS), ferricyanide,
tetramethylphenylenediamine and quinones, as well as natural
compounds such as nicotin adenine dinucleotide (NAD), nicotin
adenine dinucleotide phosphate (NADP), flavine adenine dinucleotide
(FAD), flavin mononucleotide (FMN), pyrrolo-quinoline quinone (PQQ)
and cytochromes. However, among them, FMN, PQQ, cytochromes, DCIP,
PMS, ferricyanide, tetramethylphenylenediamine and quinones are
preferable, and DCIP and/or PMS are further preferable.
[0081] The dehydrogenase of the present invention may be obtained
from any organism, but ones derived from microorganisms such as
bacteria, actinomycetes and filamentous fungi are preferable, ones
from actinomycetes are more preferable, and ones from Streptomyces
albulus are even more preferable.
[0082] The dehydrogenase from Streptomyces albulus has the
following physicochemical properties: [0083] (i) Function: The
dehydrogenase from Streptomyces albulus acts to convert a
carbon-carbon single bond on the position 3 or 6 into a
carbon-carbon double bond. [0084] (ii) Substrate specificity: The
dehydrogenase from Streptomyces albulus converts phenylahistin into
dehydrophenylahistin, and converts cyclophenylalanylhistidyl into
dehydrocyclophenylalanylhistidyl or
tetradehydrocyclophenylalanylhistidyl. [0085] (iii) Optimum pH: 8.3
[0086] (iv) pH stability: stable at 7.0-9.0 [0087] (v) Optimum
temperature: 60.degree. C. [0088] (vi) Heat stability: stable at
20-70.degree. C., deactivated at 80.degree. C. [0089] (vii)
Molecular weight: 700 kDa-800 kDa
[0090] The dehydrogenase of the present invention may be used not
only as a natural tissue or cell, but also as a cell-free extract
or enzyme solution obtained by partially or fully purifying the
cell-free extract. The dehydrogenase may be purified according to
the common enzyme purification method. Also, the multi-step
reactions may be carried out at one time by mixing other
enzymes.
[0091] The dehydrogenase of the present invention can produce a
compound wherein, in the above formula (I), at least one of (B1)
and (B2) is a carbon-carbon double bond or a compound wherein, in
the above formula (II), (B2) is a carbon-carbon double bond, by
using as a substrate a compound wherein, in the above formula (I),
at least one of (B1) and (B2) is a carbon-carbon single bond, or a
compound wherein, in the above formula (II), (B2) is a
carbon-carbon single bond. And these compounds are useful as a cell
division inhibitor or an antitumor agent.
[0092] Some examples are provided below to describe the present
invention more specifically.
[0093] The active ingredient of the cell division inhibitor of the
present invention is a substance wherein, in the above formula (I),
at least one of (B1) and (B2) is a carbon-carbon double bond, and
representative examples include substituted or non-substituted
dehydrodiketopiperazines, substituted or non-substituted
tetradehydrodiketopiperazines, substituted or unsubstituted
dehydro-cyclic dipeptide, substituted or unsubstituted
tetradehydro-cyclic dipeptide, particularly substituted or
unsubstituted dehydrocyclophenylalanylhistidyl or
tetradehydrocyclophenylalanylhistidyl represented by the above
formula (II), and further particularly dehydrophenylahistin.
[0094] As an example, the method of producing dehydrophenylahistin
is provided below, but needless to say, the present invention is
not limited thereto.
[0095] The method of collecting a novel compound
dehydrophenylahistin by culturing an actinomycete, for example,
Streptomyces albulus KO23 (which was deposited with the National
Institute of Bioscience and Human-Technology, Agency of Industrial
Science and Technology (Higashi 1-1-3, Tsukuba-shi, Ibaragi-ken,
Japan) under accession No. FERM BP-6994 on Jan. 14, 2000),
preparing a dehydrogenase from the culture, and allowing it to act
on phenylahistin is specifically described later. But, as a
dehydrogenase, either a purified enzyme or a natural cell extract
may be used. Generally, the dehydrogenase can be prepared according
to the method of culturing an actinomycete belonging to the genus
Streptomyces. After the culture, in order to purify the
dehydrogenase of the present invention from the culture solution or
prepare a cell extract containing the enzyme activity, generally a
common method used to purify the enzyme derived from microorganism
can be applied as appropriate. For example, methods such as
ultrasonic disintegration, centrifugation, salting out, dialysis,
various ion exchange resin methods, nonionic adsorption method,
chromatography including gel filtration chromatography, high
performance liquid chromatography, crystallization or freeze-drying
can be applied separately, in combination as appropriate, or
repeatedly.
[0096] The method of carrying out dehydrogenation reaction by using
an enzyme solution or cell extract prepared as above is
specifically described in the Examples later, but the fact is that
an enzyme solution and a substrate phenylahistin thereof are mixed
to react in buffer such as a phosphate buffer. If necessary, it is
possible to add an organic solvent to the reaction solution.
[0097] In order to purify and isolate dehydrophenylahistin from the
above reaction solution, generally a common method of
isolating/purifing organic compounds is applied as appropriate. For
example, methods such as various ion exchange resin methods and
nonionic adsorption methods; gel filtration chromatography,
chromatography with adsorbents including activated carbon, alumina,
silica gel etc., and high performance liquid chromatography;
crystallization; vacuum concentration; or freeze-drying can be
applied separately, in combination as appropriate, or
repeatedly.
[0098] Dehydrophenylahistin produced by the above method has cell
division inhibitory activity, as disclosed in Examples later. The
usage, dosage form and applied dose (usage) of the cell division
inhibitor of the present invention comprising dehydrophenylahistin
as an active ingredient are determined as appropriate depending on
the intended use. For example, in the case of the antitumor agent
of the present invention comprising dehydrophenylahistin as an
active ingredient, it may be administered either orally or
parenterally. Examples of the dosage forms include oral
preparations such as a tablet, powder, capsule, granule, extract
and syrup, or parenteral preparations such as an injection or
suppository. These formulations are produced using pharmaceutically
acceptable additives such as an excipient or binder according to
known methods. The applied dose of the antitumor agent containing
the above dehydrophenylahistin as an active ingredient depends on
the age, body weight, susceptibility, and symptoms of a patient.
However, the effective amount is generally about 0.1 mg to 1 g per
day per adult, and it is also possible to administer just once per
day or devidedly several times per day. Furthermore, a dose beyond
the above normal limits may be also administered as needed.
[0099] When the agent is used as a reagent for a biochemical
examination, the development of the cell cycle is inhibited at the
G2/M period, if the agent is dissolved in an organic solvent or
hydrous organic solvent and administered directly to various
cultured cell systems. Examples of the applicable organic solvents
include methanol, dimethylsulfoxide etc. Examples of the dosage
forms include solid agents such as powder or granule, liquid agents
dissolved in organic solvent or hydrous organic solvent, and the
like. Generally, an effective amount of the cell division inhibitor
comprising the above dehydrophenylahistin as an active ingredient
is 0.01-100 .mu.g/mL, but the appropriate amount depends on the
type of cultured cell system or intended use. Further, an amount
beyond the above normal limits may be also administered as
needed.
[0100] This specification includes part or all of the contents as
disclosed in the specification of Japanese Patent Application No.
2000-9370 which is a priority document of the present
application.
BEST MODE FOR CARRYING OUT THE INVENTION
[0101] The present invention will be further described in the
following examples. In the following examples,
cyclo(A.sub.1-A.sub.2), which is a cyclic dipeptide formed by
condensation of two amino acids A.sub.1 and A.sub.2 into a
diketopiperazine ring, is designated CA.sub.1A.sub.2 (A.sub.1 and
A.sub.2 represent amino acids in single-letter notation,
respectively). All of the cyclic dipeptides CA.sub.1A.sub.2 are
LL-isomers unless otherwise specified. A D-amino acid is
designated, for example, DA.sub.1, if necessary. Further,
dehydro-peptides are designated .DELTA., so that
C.DELTA.A.sub.1A.sub.2 represents cyclo(.DELTA.A.sub.1-A.sub.2),
CA.sub.1.DELTA.A.sub.2 represents cyclo(A.sub.1-.DELTA.A.sub.2),
C.DELTA.A.sub.1.DELTA.A.sub.2 represents
cyclo(.DELTA.A.sub.1-.DELTA.A.sub.2), and .DELTA.CA.sub.1A.sub.2
represents a mixture of C.DELTA.A.sub.1A.sub.2,
CA.sub.1.DELTA.A.sub.2 and C.DELTA.A.sub.1.DELTA.A.sub.2.
Furthermore, PLH represents phenylahistin.
EXAMPLE 1
(1) Phenylahistin was Prepared as Follows.
[0102] Phenylahistin-producing bacterial cells (Aspergillus ustus
NSC-F038, which was deposited with the National Institute of
Bioscience and Human-Technology, Agency of Industrial Science and
Technology (Higashi 1-1-3, Tsukuba-shi, Ibaragi-ken, Japan) under
accession No. FERM P-15830 on Sep. 3, 1996), were inoculated onto
five spots on a solid medium (20 ml per 9 cm dish) which contains
0.5% glucose, 2% glycerol, 0.2% yeast extract, 2% Pharmamedia
(cottonseed cake), 0.25% sodium chloride and 1.5% agar (pH 6.5).
The cells were then cultured at 26.degree. C. for 7 days in the
dark to obtain a spore suspension. The resulting spore suspension
(0.1 ml) was inoculated onto each of 400 dishes containing 20 ml of
the above solid medium, and then cultured at 26.degree. C. for 8
days in the dark. The resulting culture was crushed using a mixer,
and after addition of 8 L ethyl acetate, was allowed to stand for 2
days then extracted. The collected ethyl acetate layer was
concentrated under vacuum to obtain 15 g brown syrup. This syrup
was dissolved in 20 ml ethyl acetate and applied to a silica gel
column (8 cm in diameter, 20 cm in length) prepared with 1:6
acetone-ethyl acetate, followed by elution with 1:6 acetone-ethyl
acetate. The eluted solution was fractionated into 500 ml fractions
in order of elution. Phenylahistin was contained in the fifth to
tenth fractions, which were then concentrated under vacuum to
obtain 4.7 g dark brown powder in total. This dark brown powder was
dissolved in 10 ml chloroform and applied to a silica gel column (4
cm in diameter, 30 cm in length) prepared with chloroform, followed
by elution with 500 ml chloroform and then 50:1
chloroform-methanol. The compound of interest was eluted with 50:1
chloroform-methanol to obtain 1.05 g brown powder in total. After
addition of 100 ml ethyl acetate, this brown powder was mixed well
and allowed to stand for 2 days to separate out 628 mg
phenylahistin as white powder.
[0103] (2) Culture of Streptomyces albulus KO23 and preparation of
a cell-free extract were carried out as follows.
[0104] Ten milliliters of sterilized water containing 50-200 .mu.l
surfactant (Triton X-100) was added to and mixed with a slant on
which gray spores had formed well, thereby obtaining a spore
suspension. This suspension was diluted 1000-fold in a culture
medium and cultured under the following conditions. The culture
medium had the composition shown in Table 1. TABLE-US-00001 TABLE 1
KP medium composition (g/L) Glucose 15 Glycerol 10 Polypepton 10
Beef extract 10 CaCO.sub.3 4 pH 7.3
[0105] Table 2 shows the culture conditions. TABLE-US-00002 TABLE 2
Culture conditions Pre-culture in 200 ml Erlenmeyer flask KP medium
40 ml Culture period 24 hours Rotation speed 180 rpm Temperature
28.degree. C. Main culture in 5 L jar fermenter KP medium 3 L
Antifoaming agent 10 g per 3 L (Antiform AFI emulsion) Culture
period 48 hours Rotation speed 300 rpm Ventilation volume 2 L per 3
min Temperature 28.degree. C.
[0106] The cell-free extract was prepared as follows.
[0107] The culture solution (40 ml) was centrifuged at
20,000.times.g for 15 min at 4.degree. C. to collect the cells.
These cells were suspended in 40 ml physiological saline, and then
centrifuged again at 20,000.times.g for 15 min at 4.degree. C. to
wash the cells. These cells were suspended in 7.3 ml sodium
phosphate buffer (10 mM, pH 8.0), followed by ultrasonication (150
W, 1.5 min, KUBOTA INSONATOR 201M). The resulting solution was
centrifuged at 20,000.times.g for 15 min at 4.degree. C. to obtain
the supernatant as a cell-free extract.
(3) Conversion Reaction of Phenylahistin Into Dehydrophenylahistin
and Purification of the Reaction Product Were Carried Out as
Follows.
[0108] The reaction mixture had the composition shown in Table 3.
TABLE-US-00003 TABLE 3 Reaction mixture composition Phenylahistin
0.5 mg/ml Dimethyl sulfoxide 10% (v/v) Sodium phosphate buffer (pH
8.0) 9 mM Cell-free extract 0.145 units/ml Temperature 50.degree.
C.
[0109] The above reaction mixture (100 .mu.m) was divided into 200
ml Erlenmeyer flasks to contain 20 ml reaction mixture in each
flask. The reaction was carried out at 160 strokes/min for 24
hours, followed by centrifugation at 20,000.times.g for 15 min at
4.degree. C. to obtain a yellow precipitate. This precipitate was
dissolved in 55 ml methanol, and then centrifuged again at
20,000.times.g for 15 min at 4.degree. C. The resulting supernatant
was vacuumed-concentrated and dried to a solid, followed by
recrystallization from methanol, thereby obtaining 5.58 mg
dehydrophenylahistin as a yellow needle crystal.
[0110] The resulting dehydrophenylahistin has the following
physicochemical data:
[0111] EIMS m/z: 348 (M.sup.+, 100), 133 (25), 160 (17), 260
(16).
[0112] UV (MeOH) Imax, nm (e): 205 (16600), 363 (35300).
[0113] .sup.1H-NMR (500 MHz, CDCl.sub.3): .delta. 1.51, 6H, s
.delta. 5.16, 1H, d (J=17.4) .delta. 5.20, 1H, d (J=10.7) .delta.
6.03, 1H, dd (J=10.7, 17.4) .delta. 6.96, 1H, s .delta. 6.98, 1H, s
.delta. 7.32, 1H, d (J=7.0) .delta. 7.37, 2H, d (J=7.3) .delta.
7.43, 2H, dd (J=7.0, 7.3) .delta. 7.57, 1H, s .delta. 8.04, 1H, s
.delta. 9.06, 1H, br s .delta. 12.23, 1H, s
[0114] The resulting product was identified as (Z,
Z)-dehydrophenylahistin based on NOE observed between a proton of
diketopiperazine (.delta. 8.04, 1H, s) and protons of phenyl group
(d 7.43, 2H, dd (J=7.0, 7.3)). It has the following structural
formula (III): ##STR6##
EXAMPLE 2
[0115] Dehydro-products of cyclophenylalanylhistidyl (CFH) were
prepared from CFH through dehydrogenation as follows.
TABLE-US-00004 TABLE 4 Reaction mixture composition CFH 0.5 mg/ml
Dimethyl sulfoxide 10% (v/v) Sodium phosphate buffer (pH 8.0) 9 mM
Cell-free extract from Example 1 0.435 units/ml
[0116] The reaction mixture (100 ml) shown in Table 4 was prepared
and divided into five 20 ml Erlenmeyer flasks. The reaction was
carried out in Reciprocal (160 strokes/min) at 50.degree. C. for 24
hours. After 24 hours, the reaction mixture was centrifuged at
20,000.times.g for 15 min at 4.degree. C. to obtain the
supernatant. This supernatant was extracted with ethyl acetate, and
then purified by HPLC (Waters 600 Controller, 486 Tunable
Absorbance Detector, 616 Pump, Inertsil ODS-3 column .phi.20
mm.times.250 mm, 60% methanol as a solvent, flow rate of 10 ml/min,
UV detection at 256 nm), thereby obtaining three dehydro-products
at retention times of 3.9 min, 9.1 min and 11.6 min. Instrumental
analysis indicates that the product eluted at 9.1 min is
E-tetradehydrocyclophenylalanylhistidyl (CE-.DELTA.F.DELTA.H) of
the formula (IV), the product eluted at 11.6 min is
Z-tetradehydrocyclophenylalanylhistidyl (CZ-.DELTA.F.DELTA.H) of
the formula (V), and the product eluted at 30.1 min is
dehydrocyclophenylalanylhistidyl (CF.DELTA.H) of the formula (VI).
##STR7##
[0117] The compound (V) has the following physicochemical data:
[0118] EIMS m/z: 280 (M.sup.+, 100), 107 (36), 279 (29), 281
(18).
[0119] UV (MeOH) Imax, nm(e): 205 (14800), 257 (6500), 351
(27100).
[0120] .sup.1H-NMR (500 MHz, CDCl.sub.3): .delta. 6.77, 1H, s
.delta. 7.02, 1H, s .delta. 67.22, 1H, m .delta. b 7.33, 1H, t
(J=7.3) .delta. 7.37, 2H, d (J=7.3) .delta. 7.43, 2H, dd (J=7.3,
7.3) .delta. 7.75, 1H, s .delta. 8.09, 1H, s .delta. 9.30, 1H, br s
.delta. 11.91, 1H, s
EXAMPLE 3
[0121] A variety of dehydrodiketopiperazines were prepared from
different diketopiperazines as substrates through dehydrogenation
reactions using the enzyme of the present invention as follows.
TABLE-US-00005 TABLE 5 Reaction mixture composition Dimethyl
sulfoxide (DMSO) 10% (v/v) Sodium phosphate buffer (pH 8.0) 5.2 mM
Dichlorophenolindophenol (DCIP) 80 .mu.M Phenazine methosulfate
(PMS) 120 .mu.M Cell-free extract from Example 1 q.s. Substrate 0.5
mM Total 0.5 ml
[0122] The reaction mixture shown in Table 5 was used for the
dehydrogenation reaction at 37.degree. C. The reaction product was
analyzed by HPLC and detected by UV absorbance at 256 nm. This
method provided the following dehydro-products: .DELTA.CAF,
.DELTA.CFF, .DELTA.CFG, .DELTA.CFH, .DELTA.CFL, C.DELTA.FL,
CF.DELTA.L, .DELTA.CFS, .DELTA.CFV, .DELTA.CFW, .DELTA.CLW,
.DELTA.CLY, .DELTA.CVY, .DELTA.CWW, .DELTA.CWY, .DELTA.CDWY (W
residue is D-form), and .DELTA.PLH.
EXAMPLE 4
[0123] A variety of dehydrodiketopiperazines were prepared from
different diketopiperazines as substrates through dehydrogenation
reactions using the enzyme of the present invention as follows.
TABLE-US-00006 TABLE 6 Reaction mixture composition Dimethyl
sulfoxide (DMSO) 10% (v/v) Sodium phosphate buffer (pH 8.0) 5.2 mM
Cell-free extract from Example 1 q.s. Substrate 0.5 mg/ml Total 0.5
ml
[0124] The reaction mixture shown in Table 6 was used for the
dehydrogenation reaction at 37.degree. C. The reaction product was
analyzed by HPLC and detected by a photodiode array detector
(multi-channel UV, 220 nm to 400 nm). This method provided the
following dehydro-products:
[0125] .DELTA.CAH, .DELTA.CAW, .DELTA.CAY, .DELTA.CD(OMe)D(OMe),
.DELTA.CDF, .DELTA.CFG, .DELTA.CFS, .DELTA.CFV, .DELTA.CFW,
.DELTA.CGL, .DELTA.CGW, .DELTA.CGY, .DELTA.CHH, .DELTA.CHW,
.DELTA.CHY, .DELTA.CLP, .DELTA.CLW, .DELTA.CLY, .DELTA.CMM,
.DELTA.CSY, .DELTA.CVW, .DELTA.CWW, .DELTA.CWY, .DELTA.CDWY (W
residue is D-form), and .DELTA.CD(OEt)G, wherein D(OMe) represents
an aspaltic acid having a methylated carboxyl group on its side
chain (.gamma.-position), and D(OEt) represents an aspartic acid
having a ethylated carboxyl group on its side chain
(.gamma.-position).
EXAMPLE 5
[0126] A variety of dehydrodiketopiperazines were prepared from
different diketopiperazines as substrates through dehydrogenation
reactions using the enzyme of the present invention as follows.
[0127] The reaction procedures as described in Example 3 were
repeated and an amount of dehydrogenation by the enzyme was
determined based on a change in absorbance at 600 nm due to
coenzyme. Table 7 shows the amount of dehydrogenation by the enzyme
(i.e., a change in absorbance) for each substrate, which is
expressed as a relative value (an absorbance for CFL was set to
100). TABLE-US-00007 TABLE 7 Amount of dehydrogenation of each
substrate by enzyme Substrate Amount of dehydrogenation CFL 100 CFH
44 CMM 27 CEE 14 CLY 14 CDD 14
EXAMPLE 6
[0128] Dehydrogenase derived from Streptomyces albulus KO23, which
requires diketopiperazine as its substrate, was purified according
to the procedures as described in Example 1.
[0129] Streptomyces albulus KO23 was cultured in a mini jar
containing 3 L culture medium to obtain 167.12 g of the cells. The
cell-free extract was prepared from these cells as follows.
TABLE-US-00008 TABLE 8 Preparation of cell-free extract Conversion
Protein Specific Liquid Total activity (A.sub.280) activity volume
activity (units/ml) (mg/ml) (units/mg) (ml) (units) 0.684 14.2
0.0482 382 261.3
[0130] The resulting extract was subjected to DEAE-Sephacel anion
exchange column chromatography. TABLE-US-00009 Column:
DEAE-Sephacel .phi. 2.6 cm .times. 30 cm Flow rate: 1 ml/min
Fraction size: 10 ml Sample: 113 ml cell-free extract
[0131] As a buffer, 10 mM sodium phosphate buffer (pH 8.0)
containing 0.1 mM DTT was used. After the sample was adsorbed to
the column, the column was washed with 360 ml buffer, and then
eluted stepwise with 400 ml buffer containing 0.1 M NaCl, 410 ml
buffer containing 0.3 M NaCl, and 600 ml buffer containing 0.5 M
NaCl, thereby obtaining the following active fractions.
TABLE-US-00010 TABLE 9 Purification by DEAE-Sephacel anion exchange
column chromatography Conversion Protein Specific Liquid Total
activity (A.sub.280) activity volume activity Fraction (units/ml)
(mg/ml) (units/mg) (ml) (units) 50-56 0.179 1.42 0.126 70 12.5
57-71 0.240 2.56 0.0781 152 30.4
[0132] Fractions 50-56 having a higher specific activity were
subjected to the subsequent Mono-Q column chromatography as
follows. TABLE-US-00011 Column: MonoQ HR 5/5 Flow rate: 1 ml/min
Fraction size: 0.6 ml Sample: 4 .times. 1 ml DEAE-Sephacel
fractions 50-56 diluted 2- fold with buffer
[0133] As a buffer, 10 mM sodium phosphate buffer (pH 8.0)
containing 0.1 mM DTT was used. After the sample was adsorbed to
the column, the column was washed with the buffer for 4 minutes,
and then eluted with 1 M NaCl-containing buffer using a linear
gradient (25 min). The above procedures were repeated four times to
obtain the following active fraction. TABLE-US-00012 TABLE 10
Purification by MonoQ anion exchange column chromatography
Conversion Protein Specific Liquid Total activity (A.sub.280)
activity volume activity (units/ml) (mg/ml) (units/mg) (ml) (units)
0.0201 0.0376 0.646 7.2 0.145
[0134] The above active fraction was subjected to gel filtration
chromatography (Superose 12) as follows. TABLE-US-00013 Column:
Superose 12 HR 10/30 Flow rate: 0.5 ml/min Fraction size: 0.25 ml
Sample: MonoQ active fraction concentrated to 225 .mu.l
[0135] As a buffer, 10 mM sodium phosphate buffer (pH 8.0)
containing 0.1 mM DTT and 0.3 M NaCl was used. Table 11 shows
enzyme activity of each fraction. The most active fractions 13-16
were combined together and concentrated by ultrafiltration.
TABLE-US-00014 TABLE 11 Purification by Superose 12 gel filtration
column chromatography Conversion Liquid Total activity volume
activity Fraction (units/ml) (.mu.L) (units) 11, 12 0.0737 100 7.37
13, 14 0.253 45 11.4 15, 16 0.184 45 8.28 17, 18 0.0526 80 4.21
[0136] The above active fraction was subjected to gel filtration
chromatography (TSK G3000SWXL) with Waters LC Modulel as follows.
TABLE-US-00015 Column: TSK GEL G3000SWXL Flow rate: 0.5 ml/min
Sample: Superose active fraction concentrated to 40 .mu.l
[0137] As a buffer, 100 mM sodium phosphate buffer (pH 7.5)
containing 0.1 mM DTT and 0.3 M NaCl was used. The resulting active
fractions were combined together and concentrated by
ultrafiltration. Table 12 shows enzyme activity of the combined and
concentrated fraction. TABLE-US-00016 TABLE 12 Purification by TSK
G3000SWXL gel filtration chromatography Activity Protein(A.sub.280)
Specific activity (units) (mg/ml) (units/mg) 0.00224 0.00114
19.6
[0138] Table 13 shows enzyme activity in each step of the
purification procedures and a final enzyme activity. TABLE-US-00017
TABLE 13 Enzyme purification and specific activity Enzyme activity
Protein Specific activity Purification step (units) (mg) (units/mg)
Cell-free extract 0.734 15.2 0.0482 DEAE-Sephacel 0.119 0.946 0.126
Mono-Q 0.0644 0.120 0.537 Superose 12 0.00790 0.00799 0.989 TSK
G3000SW 0.00224 0.000114 19.6
EXAMPLE 7
[0139] The reaction mixture shown in Table 14 was used for the
enzymatic reaction using the enzyme of the present invention.
Various diketopiperazines were used as substrates. The resulting
enzymatic reaction mixture was tested for its inhibitory activity
against embryo division of Temnopleurus toreumaticus without any
purification of the reaction product. The test was carried out as
described in The Journal of Antibiotics, Vol. 52, p. 1017 (1999).
However, stages at which the first cleavage division occurs vary
among sea urchins, so that inhibition of the cleavage division was
observed after one hour of fertilization in this test using
Temnopleurus toreumaticus. Concentration of the substrate added to
the enzymatic reaction system was used as a criterion for inhibitor
concentration because the reaction product was used for the test
without any purification. The inhibition test for the embryo
division of Temnopleurus toreumaticus started with the highest
substrate concentration of 25 .mu.g/ml, followed by serially
diluted substrate concentrations. Table 15 shows the test results.
TABLE-US-00018 TABLE 14 Reaction mixture composition Dimethyl
sulfoxide (DMSO) 10% (v/v) Sodium phosphate buffer (pH 8.0) 5.2 mM
Cell-free extract from Example 1 q.s. Substrate 0.5 mg/ml Total 0.2
ml
[0140] TABLE-US-00019 TABLE 15 Inhibition test for cleavage
division using the enzymatic reaction mixture MIC (.mu.g/ml) CDF
reaction product >25 (80% inhibition at 25 .mu.g/ml) CFF
reaction product >25 (90% inhibition at 25 .mu.g/ml) CFV
reaction product 25 CGL reaction product >13 (70% inhibition at
13 .mu.g/ml) CHW reaction product 13 CLY reaction product >13
(60% inhibition at 13 .mu.g/ml) CWY reaction product 6.3
EXAMPLE 8
[0141] Physiological activity of each dehydrodiketopiperazine will
be described below. Each dehydrodiketopiperazine was tested for its
inhibitory activity against cleavage division of Hemicentrotus
pulcherrimus, Scaphechinus mirabilis and Temnopleurus toreumaticus
as a cell division inhibitory activity. The test was carried out as
described in The Journal of Antibiotics, Vol. 52, p. 1017 (1999).
However, stages at which the first cleavage division occurs vary
among sea urchins, so that inhibition of the cleavage division was
observed after 4 hours of fertilization in the tests using
Hemicentrotus pulcherrimus and Scaphechinus mirabilis, and after
one hour of fertilization in the test using Temnopleurus
toreumaticus, respectively. Table 16 shows the test results.
TABLE-US-00020 TABLE 16 Inhibition test for cell division using
dehydrophenylahistin and related compounds MIC, .mu.g/ml
Scaphechinus Temnopleurus Hemicentrotus Compound mirabilis
toreumaticus pulcherrimus Example 1 dehydrophenylahistin 0.0061
0.0061 0.00038 Example 2 (Z,Z)-tetradehydro-CFH 1.6 1.6 0.78
Comparison 1 (-)-phenylahistin 1.6 0.2 0.39 Comparison 2
(+)-phenylahistin >13* 6.3 13 Comparison 3 albonoursin >13*
>25* 6.3 Comparison 4 CFH >25* >25* >25* *no activity
at the indicated concentration
[0142] Dehydrophenylahistin has MIC of 0.0061 .mu.g/ml for cell
division of Scaphechinus mirabilis and Temnopleurus toreumaticus,
and MIC of 0.00038 .mu.g/ml for cell division of Hemicentrotus
pulcherrimus, respectively. Dehydrophenylahistin exhibits 250-fold
to 1000-fold inhibitory activity when compared with
non-dehydrogenated (-)-phenylahistin. (Z,Z)-tetradehydro-CFH
obtained by dehydrogenation of CFH exhibits 15-fold or more
inhibitory activity when compared with CFH. In any case, a variety
of dehydrodiketopiperazines including dehydrophenylahistin and
(Z,Z)-tetradehydro-CFH were shown to have the cell division
inhibitory activity, indicating that the dehydrodiketopiperazines
are useful as cell division inhibitors and antitumor agents.
FORMULATION EXAMPLE 1
Formulation for Injection or Drip Infusion
[0143] One milligram of dehydrophenylahistin and 5 g of glucose
powder were aseptically distributed to each vial. Each vial was
sealed under an inert gas such as nitrogen or helium, and then
stored in a cool dark place. Before use, ethanol was added to each
vial to dissolve its content, followed by addition of 100 ml 0.85%
physiological saline to produce a formulation for intravenous
injection. The resulting formulation is intravenously injected or
infused in an amount of 10 to 100 ml per day depending on
symptoms.
FORMULATION EXAMPLE 2
Formulation for Injection or Drip Infusion
[0144] The procedures as described in Formulation example 1 were
repeated to produce a formulation for intravenous injection
containing 0.2 mg dehydrophenylahistin, which may be used for
treatment of mild cases. The resulting formulation is intravenously
injected or infused in an amount of 10 to 100 ml per day depending
on symptoms.
FORMULATION EXAMPLE 3
Granules
[0145] One hundred milligrams of dehydrophenylahistin, 98 g of
lactose and 1 g of hydroxypropylcellulose were mixed well,
granulated by standard techniques, dried well and passed through a
mesh, thereby obtaining granules suitable for packaging in a bottle
or heat seal. The resulting granules are orally administered in an
amount of 100 to 1000 mg per day depending on symptoms.
[0146] All publications, patents and patent applications cited
herein are incorporated herein by reference in their entirety.
INDUSTRIAL APPLICABILITY
[0147] The present invention provides a cell division inhibitor
having stronger cell cycle inhibitory activity, particularly
antitumor activity, and an enzyme usable for the production
thereof.
* * * * *