Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Having -c- – wherein x is chalcogen – bonded directly to...
Reexamination Certificate
1999-12-15
2001-06-05
Reamer, James H. (Department: 1614)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Having -c-, wherein x is chalcogen, bonded directly to...
C549S320000
Reexamination Certificate
active
06242483
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the isolation, identification, and use of natural products. More particularly this invention is directed to substantially pure forms of cytotoxic Annonaceous acetogenins and the use of those compounds in preparing chemotherapeutic compositions.
BACKGROUND AND SUMMARY OF THE INVENTION
Annonaceous acetogenins are a well established class of natural compounds that have been isolated from plants in the Annonaceae family. It has been reported that various members of this class of compounds exhibit significant bioactivities. Acetogenins are C
35
-C
39
compounds that typically contain two long hydrocarbon chains, one of which connects a terminal 2,4-disubstituted-&ggr;-lactone to a variable number of tetrahydrofuran (THF) rings. The hydrocarbon chains contain a number of oxygenated moieties which can be hydroxyls, acetoxyls and/or ketones. Recently, single-ring acetogenins containing double bonds, epoxide compounds which lack THF rings and a compound lacking both epoxides and THF rings have been reported. These interesting newer compounds support the proposed polyketide origin of the Annonaceous acetogenins and provide additional clues to their biogenesis.
All acetogenins found to date contain multiple stereocenters, the elucidation of which often presents daunting stereochemical problems. Because of their waxy nature, the acetogenins do not produce crystals suitable for X-ray crystallographic analysis. Relative stereochemistries of ring junctions have typically been determined by comparison of natural compounds with synthetic model compounds and such methods have proven to be invaluable with the acetogenins. Recently, the absolute stereochemistries of the carbinol centers of acetogenins have been determined with the help of synthetic model compounds and high field nuclear magnetic resonance (NMR) analysis of their methoxyfluoromethylphenylacetic acid (MPTA) esters (Mosher esters).
Most Annonaceous acetogenins are potently bioactive, but the mode of action of these compounds was unknown until Londerhausen et al. concluded in
Pesticide Science
427-438 (1991), that they act to inhibit complex I of mitochondrial oxidative phosphorylation with an activity several times that of rotenone.
The present invention is directed to the isolation and characterization of the bioactivity of novel acetogenins. More particularly the present invention is directed to three new mono-tetrahydrofuran (THF) ring acetogenins each of which were isolated from the bark of
Annona squamosa.
These compounds each bear two hydroxyls that flank the THF ring and a carbonyl group at the C-9 position. The compounds were isolated using the brine shrimp lethality assay as a guide for the bioactivity-directed fractionation. It has been discovered that acetogenins possessing a hydroxyl flanking mono-THF system with a carbonyl in the aliphatic chain exhibit high selective cytotoxicity to various tumor cells and thus can be used to prepare chemotherapy compositions for administrations to patients having tumors.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to three novel acetogenins, mosinone A (1), mosin B (2) and mosin C (3). Each of those three compounds bears a single tetrahydrofuran (THF) ring and a carbonyl at the C-9 position. The specific stereochemistry of these three compounds is shown below, wherein “X”, “Y” and “Z” designate the stereochemistry across the THF ring.
wherein X is threo, Y is trans, Z is threo and C
15
/C
20
is R/R.
wherein, for mosin B: X is threo/erythro, Y is trans, Z is threo/erythro and C
15
/C
20
is R/S or S/R; and
for mosin C: X is threo, Y is cis, Z is threo and C
15
/C
20
is R/S.
These three compounds and one previously known acetogenin, annoreticuin-9-one (4) were isolated from the bark of
A. squamosa.
The stereochemistry of annoreticuin-9-one is as follows:
wherein X is threo, Y is trans, Z is threo and C
15
/C
20
is R/R.
(2,4-cis and trans)-Mosinone A (1) is a mixture of ketolactone compounds bearing a threo/trans/threo ring relationship and a double bond two methylene units away from the flanking hydroxyl. The other two new acetogenins differ in their stereochemistries around the THF ring; mosin B (2) has a threo/trans/erythro configuration across the ring and mosin C (3) possesses a threo/cis/threo relative stereochemistry. Annoreticuin-9-one (4), a known acetogenin which bears a threo/trans/threo ring configuration and a C-9 carbonyl and is new to this species. The structures were elucidated based on spectroscopic and chemical methods.
Each of these compounds was isolated from the bark of
A. squamosa.
Approximately 7.4 kg-of dried bark was pulverized and extracted with ethanol then further partitioned to yield 545.5 g of F005 (BST LC
50
=1.5155). From this, 500.5 g was loaded onto a silica gel (Si gel) column and eluted with hexane and increasing percentages of chloroform, then chloroform and increasing percentages of methanol. Sixty fractions were collected, and fractions 30-36 were combined (21.67 g). This material was separated on successive open columns packed with Si gel. Repeated HPLC of fractions active in the brine shrimp lethality test (BST) yielded compounds 1-4. The procedure for conducting the BST test is described in Meyer et al.
Planta Med.
45, p. 31-34 (1982).
(2,4-cis- and trans)-Mosinone A (1) was isolated in a mixture as a white waxy solid. The molecular weight of 1 was shown to be 620, based on the MH
+
peak at 621 in the CIMS. The compound was established as having the elemental composition of C
37
H
64
O
7
by the high resolution CIMS peak for the MH
+
at m/z 621.4723 (621.4730 calcd.). Signals in the
1
H NMR of 1 at &dgr; 4.39 and 4.55 (Table 1), with a combined integration for one proton, were assigned to H-4 and indicated the presence of a (2,4-cis and trans)-mixture at the ketolactone moiety which is common for acetogenins of this type. Resonances in the
1
H NMR of 1 at &dgr; 2.65 and 3.07 (H-35) and at 2.20 (H-37) further substantiated this assignment. In the
13
C NMR of 1, signals at &dgr; 205.5 (C-36), 178.7 and 178.1 (C-1), 44.2 and 43.7 (C-2), 79.0 and 78.5 (C-4), and 23.7 (Table 2) also confirmed that 1 is a cis/trans mixture of ketolactone isomers. The stereochemistry at C-4 was assumed as R, based on spectral comparisons with (2,4-cis and trans)-bullatacinone, which has known chirality, and the fact that all 4-oxygenated acetogenins known to date are 4-R.
TABLE 1
1
H NMR spectral data (&dgr;) for 1-4.
1
H NMR (500 MHz, CDCl
3
, J in Hz)
Position
1 trans
1 cis
2
3
4
1
—
—
—
—
—
2
3.02 m
3.03 m
—
—
—
3a
1.99 m
1.48 m
2.40 dddd
2.40 dddd
2.40 dddd
(15.0, 8.2,
(15.0, 8.2,
(15.0, 8.2,
1.5, 1.5)
1.5, 1.5)
1.5, 1.5)
4
4.55 dddd
4.39 dddd
3.87 m
3.86 m
3.86
(8.3, 8.2,
(10.7, 7.4,
5.7, 3.2)
5.4, 5.4)
5a
1.48 m
1.60 m
1.48 m
1.47 m
1.47 m
5b
1.56 m
1.76 m
1.48 m
1.47 m
1.47 m
6-7
1.26 br s
1.26 br s
1.26 br s
1.26 br s
1.26 br s
8
2.40 t
2.40 t
2.40 t
2.40 t
2.40 t
(7.5)
a
(7.5)
a
(7.5)
a
(7.5)
a
(7.0)
a
9
—
—
—
—
—
10
2.42 t
2.42 t
2.42 t
2.42 t
2.42 t
(7.5)
a
(7.5)
a
(7.5)
a
(7.5)
a
(7.5)
a
11-13
1.26 br s
1.26 br s
1.26 br s
1.26 br s
1.26 br s
14
1.41 m
1.41 m
1.41 m
1.41 m
1.41 m
15
3.40 m
3.40 m
3.38 m
b
3.42 m
3.40 m
16
3.80 m
3.80 m
3.81 m
c
3.81 m
3.79 m
17
1.69 m,
1.69 m,
1.97 m,
1.94 m,
1.99 m,
1.99 m
1.99 m
1.56 m
d
1.75 m
1.69 m
18
1.69 m,
1.69 m,
1.87 m,
1.94 m,
1.99 m,
1.99 m
1.99 m
1.83 m
d
1.75 m
1.69 m
19
3.80 m
3.80 m
3.82 m
c
3.81 m
3.79 m
20
3.41 m
3.41 m
3.87 m
b
3.42 m
3.40 m
21
2.00 m
2.00 m
1.41
1.41 m
1.41 m
22
2.20 m
2.20 m
1.26 br s
1.26 br s
1.26 br s
23
5.36 m
5.36 m
1.26 br s
1.26 br s
1.26 br s
24
5.39 m
5.39 m
1.26 br s
1.26 br s
1.26 br s
25
2.04 m
2.04m
1.26 br s
1.26 br s
1.26 br s
26-30
1.26 br s
1.26 br s
1.26 br s
1.26 br s
1.26 br s
31
1.29 m
1.29 m
1.30 m
1.29 m
1.29 m
32
0.88 t
0.88 t (7.0)
0.88 t
0.88 t
0.88 t
(7.0)
(7.0)
(7.0)
(7.0)
33a
2.67 dd
2.61 dd
7.19 d
7.19 d
7.19 d
(18.5, 9.0)
(18.5, 9.0)
(1.5)
(1.5)
(1.5)
33b
3.04 dd
3.11 (18.5,
—
—
—
(1
Hopp David Craig
McLaughlin Jerry L.
Barnes & Thornburg
Purdue Research Foundation
Reamer James H.
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