U.S. patent application number 10/818436 was filed with the patent office on 2004-12-16 for anti-cancer nitro- and thia-fatty acids.
Invention is credited to Easton, J. Christopher, Ferrante, Antonio, Hll, S.T. Charles.
Application Number | 20040254240 10/818436 |
Document ID | / |
Family ID | 3817079 |
Filed Date | 2004-12-16 |
United States Patent
Application |
20040254240 |
Kind Code |
A1 |
Ferrante, Antonio ; et
al. |
December 16, 2004 |
Anti-cancer nitro- and thia-fatty acids
Abstract
The present invention relates to pharmaceutical compositions
comprising, as an anticancer agent (a) one or more compounds having
the formula NO.sub.2-A-B, wherein A is a saturated or unsaturated
hydrocarbon chain of 14-26 double bonds, and B is
(CH.sub.2).sub.m(COOH).sub.n in which n is an integer from 0 to 2
and m is an integer from 0 to 2; or a derivative thereof in which
the hydrocarbon chain has one or more than one substitution
selected from the group consisting of hydroxy, hydroperoxy, epoxy
and peroxy; (b) one or more compounds selected from polyunsaturated
fatty acids (PUFA's) having a 16 to 26 carbon atom chain and 3 to 6
double bonds, and wherein the PUFA is covalently coupled at the
carboxylic acid group to an amino acid selected from glycine and
aspartic acid; (c) one or more compounds selected from unsaturated
fatty acids having an 18 to 25 carbon atom chain and 1 to 6 double
bonds and wherein the fatty acid has one or two .beta.-oxa,
.gamma.-oxa, .beta.-thia, .gamma.-thia substitutions; or (d) one or
more compounds having formula (I) wherein A' is a saturated or
unsaturated hydrocarbon chain of 9-26 carbon atoms, X is oxygen or
is absent and B' is (CH.sub.2).sub.j(COOH).sub.k in which j is an
integer from 1 to 3 and k is 0 or 1; or a derivative thereof in
which the hydrocarbon chain has one or more than one substitution
selected from the group consisting of hydroxy, hydroperoxy, epoxy
and peroxy; and a pharmaceutically acceptable carrier or diluent.
1
Inventors: |
Ferrante, Antonio; (South
Australia, AU) ; Easton, J. Christopher; (North
Adelaide, AU) ; Hll, S.T. Charles; (North Adelaide,
AU) |
Correspondence
Address: |
KLAUBER & JACKSON
411 HACKENSACK AVENUE
HACKENSACK
NJ
07601
|
Family ID: |
3817079 |
Appl. No.: |
10/818436 |
Filed: |
April 5, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10818436 |
Apr 5, 2004 |
|
|
|
10100274 |
Mar 18, 2002 |
|
|
|
Current U.S.
Class: |
514/509 |
Current CPC
Class: |
Y02A 50/411 20180101;
C07C 205/14 20130101; A61K 31/19 20130101; A61K 31/20 20130101;
A61P 39/06 20180101; A61P 37/06 20180101; A61P 33/06 20180101; A61P
11/06 20180101; A61P 11/00 20180101; A61P 17/06 20180101; A61P
43/00 20180101; A61P 33/00 20180101; A61K 31/202 20130101; A61P
9/10 20180101; C07C 205/02 20130101; C07C 205/51 20130101; A61P
1/04 20180101; A61P 9/00 20180101; A61P 37/08 20180101; C07C 45/30
20130101; C07C 205/50 20130101; C07C 409/40 20130101; A61P 29/00
20180101; A61P 3/10 20180101; A61K 31/21 20130101; A61K 31/335
20130101; A61P 1/18 20180101; A61P 11/02 20180101; A61P 35/00
20180101; A61P 37/02 20180101; C07C 205/03 20130101; C07C 205/15
20130101; A61P 31/00 20180101; C07C 17/16 20130101; C07C 323/52
20130101; Y02A 50/30 20180101; A61P 17/00 20180101; C07C 317/44
20130101; A61K 45/06 20130101; A61P 25/00 20180101; C07C 17/16
20130101; C07C 19/075 20130101; C07C 17/16 20130101; C07C 21/14
20130101; C07C 45/30 20130101; C07C 47/02 20130101; C07C 45/30
20130101; C07C 47/21 20130101; A61K 31/21 20130101; A61K 2300/00
20130101; A61K 31/335 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/509 |
International
Class: |
A61K 031/21 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 17, 1999 |
AU |
PQ 2914 |
Claims
1. An anti-cancer pharmaceutical composition comprising, as an
anti-cancer agent, one or more compounds having the formula
NO.sub.2-A-B, wherein A is a saturated or unsaturated hydrocarbon
chain of 14 to 26 carbon atoms and B is (CH.sub.2).sub.n
(COOH).sub.m in which n is an integer from 0 to 2 and m is an
integer from 0 to 2, and a pharmaceutically acceptable carrier or
diluent.
2. A pharmaceutical composition according to claim 1, in which the
hydrocarbon chain of the compound(s) includes one or more than one
substitution selected from the group consisting of hydroxy,
hydroperoxy, epoxy and peroxy.
3. A pharmaceutical composition according to claim 1, in which the
hydrocarbon chain of the compound(s) is either saturated or
unsaturated and has 18 to 22 carbon atoms.
4. A pharmaceutical composition according to claim 1, in which the
hydrocarbon chain of the compound(s) has from 3 to 6 double
bonds.
5. A pharmaceutical composition according to claim 1, in which the
hydrocarbon chain of the compound(s) is unsaturated and has
eighteen carbon atoms and three double bonds separated by methylene
groups, with the first double bond relative to the omega carbon
atom being between the 3<rd>and 4<th>or 6<th>and
7<th>carbon atoms.
6. An anti-cancer pharmaceutical composition comprising, as an
anti-cancer agent, the compound
(all-Z)-4-nitrotricosa-8,11,14,17-tetraenoic acid or
4-[(all-Z)-Nonadeca-4,7,10,13-tetraenyl]-4-nitroheptane-1,7-dicarboxylic
acid.
7. A pharmaceutical composition according to any one of claims 1 to
6, in which the cancer is prostate cancer.
8. A pharmaceutical composition according to any one of claims 1 to
6, in which the cancer is breast cancer.
9. A method of treating cancer in a subject, said method comprising
administering to the subject a therapeutic amount of a compound
having the formula NO.sub.2-A-B, wherein A is a saturated or
unsaturated hydrocarbon chain of 14 to 26 carbon atoms and B is
(CH.sub.2)n(COOH)m in which n is an integer from 0 to 2 and m is an
integer from 0 to 2.
10. A method according to claim 9, in which the hydrocarbon chain
of the compound includes one or more than one substitution selected
from the group consisting of hydroxy, hydroperoxy, epoxy and
peroxy.
11. A method according to claim 9, in which the hydrocarbon chain
of the compound is either saturated or unsaturated and has 18 to 22
carbon atoms
12. A method according to claim 9, in which the hydrocarbon chain
of the compound has from 3 to 6 double bonds.
13. A method according to claim 9, in which the hydrocarbon chain
of the compound is unsaturated and has eighteen carbon atoms and
three double bonds separated by methylene groups, with the first
double bond relative to the omega carbon atom being between the 3rd
and 4th or 6th and 7th carbon atoms.
14. A method according to claim 9, wherein the compound is
(all-Z)-4-nitrotricosa-8,11,14,17-tetraenoic acid or
4-[(all-Z)-Nonadeca-4,7,
10,13-tetraenyl]-4-nitroheptane-1,7-dicarboxylic acid.
15. A method according to any one of claims 9 to 14, in which the
cancer is prostate cancer.
16. A method according to any one of claims 9 to 14, in which the
cancer is breast cancer.
17. Use of a compound having the formula NO.sub.2-A-B, wherein A is
a saturated or unsaturated hydrocarbon chain of 14 to 26 carbon
atoms and B is (CH.sub.2).sub.n (COOH).sub.m in which n is an
integer from 0 to 2 and m is an integer from 0 to 2, for the
preparation of a pharmaceutical composition for the treatment of
cancer.
18. Use according to claim 17, in which the hydrocarbon chain of
the compound includes one or more than one substitution selected
from the group consisting of hydroxy, hydroperoxy, epoxy and
peroxy.
19. Use according to claim 17, in which the hydrocarbon chain of
the compound is either saturated or unsaturated and has 18 to 22
carbon atoms.
20. Use according to claim 17, in which the hydrocarbon chain of
the compound has from 3 to 6 double bonds.
21. Use according to claim 17, in which the hydrocarbon chain of
the compound is unsaturated and has eighteen carbon atoms and three
double bonds separated by methylene groups, with the first double
bond relative to the omega carbon atom being between the 3rd and
4th or 6th and 7th carbon atoms.
22. Use according to claim 17, wherein the compound is
(all-Z)-4-nitrotricosa-8,11,14,17-tetraenoic acid or
4-[(all-Z)-Nonadeca-4,7,
10,13-tetraenyl]-4-nitroheptane-1,7-dicarboxylic acid.
23. Use according to any one of claims 17 to 22, wherein the cancer
is prostate cancer.
24. Use according to any one of claims 17 to 22, wherein the cancer
is breast cancer.
25. An anti-cancer pharmaceutical composition comprising, as an
anti-cancer agent, one or more compounds selected from
polyunsaturated fatty acids having a 16 to 26 carbon atom chain and
3 to 6 double bonds, and wherein the polyunsaturated fatty acid is
covalently coupled at the carboxylic acid group to an amino acid
selected from glycine and aspartic acid, and a pharmaceutically
acceptable carrier or diluent.
26. A method of treating cancer in a subject, said method
comprising administering to the subject a therapeutic amount of a
compound selected from polyunsaturated fatty acids having a 16 to
26 carbon atom chain and 3 to 6 double bonds, and wherein the
polyunsaturated fatty acid is covalently coupled at the carboxylic
acid group to an amino acid selected from glycine and aspartic
acid.
27. Use of a compound selected from polyunsaturated fatty acids
having a 16 to 26 carbon atom chain and 3 to 6 double bonds, and
wherein the polyunsaturated fatty acid is covalently coupled at the
carboxylic acid group to an amino acid selected from glycine and
aspartic acid, for the preparation of a pharmaceutical composition
for the treatment of cancer.
28. An anti-cancer pharmaceutical composition comprising, as an
anti-cancer agent, one or more compounds selected from unsaturated
fatty acids having an 18 to 25 carbon atom chain and 1 to 6 double
bonds and wherein the fatty acid has one or two substitutions
selected from the group consisting of [beta]-oxa, [gamma]-oxa,
[beta]-thia and [gamma]-thia, and a pharmaceutically acceptable
carrier or diluent.
29. A method of treating cancer in a subject, said method
comprising administering to the subject a therapeutic amount of a
compound selected from unsaturated fatty acids hasting an 18 to 25
carbon atom chain and 1 to 6 double bonds and wherein the fatty
acid has one or two substitutions selected from the group
consisting of [beta]-oxa, [gamma]-oxa, [beta]-thia and
[gamma]-thia.
30. Use of a compound selected from unsaturated fatty acids having
an 18 to 25 carbon atom chain and 1 to 6 double bonds and wherein
the fatty acid has one or two substitutions selected from the group
consisting of [beta]-oxa, [gamma]-oxa, [beta]-thia and
[gamma]-thia, for the preparation of a pharmaceutical composition
for the treatment of cancer.
31. An anti-cancer pharmaceutical composition comprising, as an
anti-cancer agent, one or more compounds selected from compounds
having the formula 38wherein A is a saturated or unsaturated
hydrocarbon chain of 9 to 26 carbon atoms, X is oxygen or is absent
and U is (CH.sub.2)j(COOH)k in which j is an integer from 1 to 3
and k is 0 or 1, and derivatives thereof in which the hydrocarbon
chain includes one or more substitution selected from the group
consisting of hydroxy, hydroperoxy, epoxy and peroxy, and a
pharmaceutically acceptable carrier or diluent.
32. A method of treating cancer in a subject said method comprising
administering to the subject a therapeutic amount of a compound
having the formula 39wherein A is a saturated or unsaturated
hydrocarbon chain of 9 to 26 carbon atoms, X is oxygen or is absent
and B is (CH.sub.2).sub.j (COOH).sub.k in which j is an integer
from 1 to 3 and k is 0 or 1, or a derivative thereof in which the
hydrocarbon chain includes one or more substitution selected from
the group consisting of hydroxy, hydroperoxy, epoxy and peroxy.
33. Use of a compound selected from compounds having the formula
40wherein A is a saturated or unsaturated hydrocarbon chain of 9 to
26 carbon atoms, X is oxygen or is absent and B is
(CH.sub.2)j(COOH)k in which j is an integer from 1 to 3 and k is 0
or 1, or a derivative thereof in which the hydrocarbon chain
includes one or more substitution selected from the group
consisting of hydroxy, hydroperoxy, epoxy and peroxy, for the
preparation of a pharmaceutical composition for the treatment of
cancer.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to compounds which include a
carbon chain of 14 to 26 carbon atoms and a nitro or sulphur group.
In a particular embodiment the invention relates to nitro analogues
of polyunsaturated fatty acids. The present invention further
relates to the use of these compounds in methods of treatment.
BACKGROUND OF THE INVENTION
[0002] Fatty acids are one of the most extensively studied classes
of compounds due to their important role in biological
systems.sup.(1,2). Hundreds of different fatty acids exist in
nature. They consist of saturated, monounsaturated and
polyunsaturated fatty acids, having chain lengths from 4 to 22
carbon atoms. Polyunsaturated fatty acids (PUFAs) contain 16 to 22
carbon atoms with two or more methylene interrupted double bonds.
The PUFA, arachidonic acid, contains 20 carbons and four
methylene-interrupted cis-double bonds commencing six carbons from
the terminal methyl group, which therefore leads to an abbreviated
nomenclature of 20:4 (n-6).
[0003] PUFAs can be divided into four families, based on the parent
fatty acids from which they are derived: linoleic acid (18:2 n-6),
.alpha.-linolenic acid (18:3 n-3), oleic acid (18:1 n-9) and
palmitoleic acid (16:1 n-7). The n-6 and n-3 PUFAs cannot be
synthesised by mammals and are known as essential fatty acids
(EFAs). They are required by mammalian bodies indirectly through
desaturation or elongation of linoleic and .alpha.-linolenic acids,
which must be supplied in the diet.
[0004] EFAs have a variety of biological activities. For instance,
it has been suggested that they are important modulators of
neoplastic development because they are capable of decreasing the
size and number of tumours as well as the lagtime of tumour
appearance..sup.[3] Intake of n-3 PUFAs has been found to be
associated with a reduced incidence of coronary arterial diseases,
and various mechanisms by which n-3 PUFAs act have been
proposed..sup.[4,5] Some n-3 and n-6 PUFAs also possess
antimalarial.sup.[6] or anti-inflammatory properties..sup.[7]
Furthermore, one of the EFAs' most important biological roles is to
supply precursors for the production of bioactive fatty acid
metabolites that can modulate many immune functions..sup.[8]
[0005] Arachidonic acid (AA) is the most extensively studied of the
EFAs and it is a principal precursor for many important biological
mediators. There are two pathways for arachidonic acid metabolism
(1) the cycloxygenase pathway which leads to the formation of
prostaglandins and thromboxanes, and (2) the lipoxygenase pathway
which is responsible for the generation of leukotrienes and
lipoxins. These metabolites, collectively called eicosanoids, have
been implicated in the pathology of a variety of diseases such as
asthma.sup.[9] and other inflammatory disorders..sup.[10,11]
[0006] Although EFAs play important roles in the biological process
of the mammalian body, they are not widely used as therapeutics due
to their limited availability in vitro. They are readily degradable
by .beta.-oxidation, which is the major oxidative pathway in fatty
acid metabolism. The net process of .beta.-oxidation is
characterised by the degradation of the fatty acid carbon chain by
two carbon atoms with the concomitant production of equimolar
amounts of acetyl-coenzyme A.
[0007] To overcome the problem of .beta.-oxidation, some work has
been done to design and synthesise modified PUFAs, such as the
.beta.-oxa and .beta.-thia PUFAs.sup.[12,13]. These compounds were
shown to have enhanced resistance to .beta.-oxidation while still
retaining certain biological activities of the native PUFAs.
[0008] The present invention relates to another group of modified
PUFAs, the nitro analogues of PUFAs. The rationale was that the
nitro group is chemically similar to COOH group with regard to
size, charge and shape. In addition, the nitro compounds are a
group of relatively stable compounds and are resistant to
.beta.-oxidation by preventing CoA thioester production, which is
the first step in oxidation of fatty acids. This also means that
the nitro compounds will not be incorporated into lipids and will
more likely be present in a free form.
SUMMARY OF THE PRESENT INVENTION
[0009] A first group of compounds of the present invention have the
general formula I:-- 2
[0010] in which A is a saturated or unsaturated hydrocarbon chain
of 14 to 26 carbon atoms; and B is (CH.sub.2).sub.n(COOH).sub.m in
which n is 0 to 2 and m is 0 to 2;
[0011] and the derivatives thereof having a further one or more
than one substitution selected from the group consisting of
hydroxy, hydroperoxy, epoxy and peroxy.
[0012] In a preferred embodiment of the present invention, A is a
hydrocarbon chain of 18 to 22 carbon atoms which is preferably
polyunsaturated, and in particular has 3-6 double bonds.
[0013] More preferably, the compound has an unsaturated hydrocarbon
chain having 18 carbon atoms and three double bonds separated by
methylene groups, with the first double bond relative to the omega
carbon atom being between the 3.sup.rd and 4.sup.th or 6.sup.th and
7.sup.th carbon atoms.
[0014] In a further preferred embodiment, the compound is selected
from the group consisting of those set out in Table 1.
1TABLE 1 Structure and nomenclature of nitro fatty acid analogues
Structure Systematic Name WCH Report Thesis 3 1-Nitrooctadecane Lx1
4a 55 4 (z,z,z)-1-Nitro-9,12,15-octadecatriene Lx2 4c 60a 5
(z,z,z)-1-Nitro-6,9,12-octadecatriene Lx3 4d 60b 6
(all-z)-1-Nitro-5,8,11,14-eico- satetraene Lx4 4b 60c 7
(all-z)-1-Nitro-4,7,10,13,16,19-docosa- hexaene Lx5 4e 60 8
4-Nitrohenicosanoic acid Lx6 6a 80 9
(all-Z)-4-Nitrotricosa-8,11,14,17-tetra- enoic acid Lx7 6b 82 10
3-Heptadecyl-3-nitropentane-1,5-di- carboxylic acid Lx8 8a 84 11
3-[(all-Z)-Nonadeca-4,7,10,13-tetraenyl]-3-nitro-
pentane-1,5-dicarboxylic acid Lx9 8b 86
[0015] In yet a further preferred embodiment, the compound is Lx2
or Lx3.
[0016] In yet a further preferred embodiment, the compound is Lx7
or Lx9.
[0017] The ability of the compounds of the present invention to
inhibit lipoxygenase activity suggests their use in the treatment
of cancer, eg prostate cancer.
[0018] They may also find application in the treatment of cancer eg
prostate cancer.
[0019] The metabolism of arachidonic acid has been a topic of great
interest, particularly in relation to its role in inflammation. A
major interest has been the search for selective inhibitors of the
various enzymes in the arachidonic acid cascade. This is critical
for the development of compounds with therapeutic potential for
control of the pathological processes mediated by arachidonic acid
metabolites, and is also important in providing useful biochemical
tools for mechanistic investigation of the enzymes involved.
Considerable effort in this area has been made in association with
the cycloxygenase pathway, and a number of nonsteroidal
anti-inflammatory drugs (e.g. aspirin and indomethacin) have been
found to have inhibitory effects on cycloxygenase.[.sup.14] More
recently, efforts have been extended to a study of the lipoxygenase
(LO) pathway and the search for selective inhibitors of the enzymes
involved in the pathway. Another major objective of the present
work is to assess the possible activity for enzyme inhibition or
other potential physiological activities of the synthetic nitro
compounds using enzymological and biological assays.
[0020] In a first aspect, the present invention consists in an
anti-cancer pharmaceutical composition comprising at least one
compound of formula I and a pharmaceutically acceptable carrier or
diluent.
[0021] In a second aspect, the present invention consists in a
method of treating cancer (eg prostate cancer) in a subject, the
method comprising administering to the subject a therapeutic amount
of a compound of formula I.
[0022] In a third aspect, the present invention consists in an
anti-cancer pharmaceutical composition comprising at least one
compound being a polyunsaturated fatty acid (PUFA) having a 16-26
carbon atom chain and 3-6 double bonds, and wherein the PUFA is
covalently coupled at the carboxylic acid group to an amino acid
selected from glycine and aspartic acid. These PUFA analogues are
described in International Patent Specification No.
PCT/AU95/00717.
[0023] In a fourth aspect, the present invention consists in a
method of treating cancer in a subject, the method comprising
administering to the subject a therapeutic amount of such a PUFA
analogue.
[0024] In a fifth aspect, the present invention consists in an
anti-cancer pharmaceutical composition comprising at least one
compound being a PUFA having an 18-25 carbon atom chain and 1-6
double bonds and wherein the PUFA has one or two substitutions
selected from the group consisting of .beta.-oxa, .gamma.-oxa,
.beta.-thia and .gamma.-thia. These compounds are described in
International Patent Specification No. PCT/AU95/00677.
[0025] In a sixth aspect, the present invention consists in a
method of treating cancer in a subject, the method comprising
administering to the subject a therapeutic amount of such an oxa-
or thia-fatty acid.
[0026] In a seventh aspect, the present invention consists in an
anti-cancer pharmaceutical composition comprising at least one
compound of the formula 12
[0027] wherein A is a saturated or unsaturated hydrocarbon chain of
9-26 carbon atoms, X is oxygen or is absent and B is
(CH.sub.2).sub.j(COOH).su- b.k in which j is an integer from 1 to 3
and k is 0 or 1; or a derivative thereof in which the hydrocarbon
chain includes one or more than one substitution selected from the
group consisting of hydroxy, hydroperoxy, epoxy and peroxy.
[0028] In an eighth aspect, the present invention relates to a
method of treating cancer in a subject, the method comprising
administering to the subject a therapeutic amount of such a thia or
sulfinyl fatty acid.
[0029] In order that the nature of the present invention may be
more clearly understood, preferred forms thereof will now be
described with reference to the following examples.
[0030] A. Preparation of Nitro Analogues of PUFA
[0031] (1) Synthesis of nitroalkanes/nitroalkenes (Lx1 to Lx5)
[0032] The first target compounds were a series of nitro compounds
with chain lengths of 18 to 22 carbons and 3 to 5 double bonds,
being prepared by modification of commercially available
polyunsaturated fatty alcohols. Since the unsaturated alcohols are
relatively expensive to obtain, stearyl alcohol was used as the
starting material for establishing synthetic methods.
[0033] The synthesis of nitroalkanes/nitroalkenes.sup.[15] Lx1 to
Lx5 is summarised in Scheme 1. 13
[0034] Stearyl alcohol 1a was converted to stearyl bromide 2a by
treatment with triphenyl phosphine (PPh.sub.3) and carbon
tetrabromide (CBr.sub.4) in dichloromethane overnight at room
temperature. After purification by flash chromatography on silica
gel, stearyl bromide 2a was obtained in 96% yield. Treatment at the
stearyl bromide with silver nitrate in ether afforded stearyl
nitrate 4a in low yield (<10%). Attempts to improve the yield of
the nitroalkane 4a from this procedure by extending reaction time
and increasing the amount of silver nitrate used were unsuccessful
and so conversion of the bromide to the nitroalkane via the iodide
was investigated. Conversion of stearyl bromide 2a to the
corresponding iodide 3a was achieved in the yields of >90% as
estimated by the .sup.1H NMR spectrum of crude reaction mixture.
Stearyl iodide 3a was converted in situ to stearyl nitrate 4a, by
treatment with silver nitrate in ether for 3 days at room
temperature, and the product, stearyl nitrate 4a, was obtained in
65% yield. Based on this approach, nitroalkenes 4b-4e were
synthesised and fully characterised (Scheme 1).
[0035] (2) Synthesis of .gamma.-nitroalkanoic and
.gamma.-nitroalkenoic Acids [6a (Lx6) and 6b (Lx7)]
[0036] The synthetic nitroalkane and nitroalkene (Lx1 and Lx4) were
further used as starting material for synthesis of
.gamma.-nitroalkanoic and .gamma.-nitroalkenoic acids (Lx6 and
Lx7). The .gamma.-nitroalkanoic and .gamma.-nitroalkenoic acid
esters 5a and 5b were produced by Michael addition of the
respective nitroalkane and nitroalkene 4a and 4b to methyl
acrylate. The esters were then hydrolysed to give the
.gamma.-nitroalkanoic and .gamma.-nitroalkenoic acids 6a and 6b
(Scheme 2): 14
[0037] A published method.sup.[16] for the synthesis of short chain
.gamma.-nitroalkanoic acid esters was investigated for synthesis of
the long chain acid ester 5a. The nitroalkane 4a was treated with
methyl acrylate in a two phase system of water and dichloromethane
in the presence of sodium hydroxide at room temperature for 24
hours. No reaction occurred under these conditions and a
modification was then made where tetrabutylammonium iodide (TBAI),
a phase transfer catalyst, was introduced into the reaction to
improve the solubility of the base in the organic phase. With this
change, a small amount of the expected product was detected by
.sup.1H NMR analysis of the crude reaction residue. The yield of
.gamma.-nitroalkanoic acid ester 5a was further improved (reaching
69% yield) by increasing the relative amount to 3:1 (for methyl
acrylate: nitroalkane) and by increasing the reaction temperature
to 5.degree. C. The .gamma.-nitroalkanoic acid ester 5a was
hydrolysed by treatment with either 1.5M lithium hydroxide in
dimethoxyethane (DME) or aluminium tribromide in
tetrahydrothiophene (Th) at room temperature to afford the
.gamma.-nitroalkanoic acid 6a in 98% yield. The unsaturated
nitroalkenoic acid 6b was generated in similar yield using the same
method, and both 6a and 6b were fully characterised.
[0038] (3) Synthesis of .alpha.,.alpha.-dipropanate Nitroalkane and
Nitroalkene [8a (Lx8) and 8b (Lx9)]
[0039] Multiple Michael addition to primary nitroalkanes can lead
to the production of multiply substituted nitroalkanes..sup.[7]
Based on this, the .alpha.,.alpha.-dipropanate ester nitroalkane
and nitroalkene 7a and 7b were prepared by Michael addition of the
nitroalkane and nitroalkene 4a and 4b to methyl acrylate in the
presence of 1,8-diazabicyclo [5,4,0] undec-7-ene (DBU) as a strong
base. The resulting diesters 7a and 7b were converted to the
corresponding dicarboxylic acids 8a and 8b by lithium hydroxide
hydrolysis (78-80% yield) (Scheme 3): 15
[0040] (4) Synthesis of .alpha.,.beta.-unsaturated nitroalkenes
(11a and 11b) The reaction scheme shown below (Scheme 4) was
envisaged for generation of .alpha.,.beta.-unsaturated
nitroalkenes. 16
[0041] Fatty alcohol 1a was oxidised by pyridinium chlorochromate
(PCC) in dichloromethane at room temperature to yield corresponding
aldehyde 9a..sup.[18] .beta.-hydroxy nitroalkane can be efficiently
obtained by nitroaldol reaction,.sup.[19] and in this case, the
aldehyde 9a reacted with nitromethane in ether, with Amberlyst A-21
as a heterogeneous basic catalyst, generating the .beta.-hydroxy
nitroalkanes in 89% yield after purification. Dehydration of
.beta.-hydroxy nitroalkane 10a.sup.[20] was undertaken by mixing
with 1 equivalent of methanesulfonyl chloride (CH.sub.3SO.sub.2Cl)
and 4 equivalents of triethylamine in dry dichloromethane at
0.degree. C. The .sup.1H NMR spectrum of the residue indicated that
the products were a mixture of conjugated and nonconjugated nitro
compounds. In subsequent experiments, this reaction was monitored
by TLC from 5 mins to 2.5 hours. The result showed that only the
conjugated product 11a could be seen at 5 mins, and after 10 mins
of reaction, the nonconjugated product 12a showed up and it became
predominant after 2 hrs reaction. Although conjugated 11a and
nonconjugated nitro compound 12a were distinguishable by .sup.1H
NMR and .sup.13C NMR, and were separable by TLC, no pure samples of
either compound were obtained by flash chromatography due to
decomposition. A similar result was obtained for synthesis of
conjugated compound 11b.
[0042] The variation in the product distribution (11a and 12a)
during reaction may be explained on the basis of kinetic versus
thermodynamic control. It is possible that the nonconjugated
compound 12a is thermodynamically more stable, but the formation of
the conjugated product 11a is kinetically favoured over that of the
nonconjugated product 12a. However, once the reaction for
conjugated compound formation reached a kinetic equilibrium,
formation of the nonconjugated compound will become predominant
because of its higher thermodynamic stability. However, further
work is needed to elucidate this.
[0043] (5) Synthesis of .alpha.-Nitro Acids 13a
[0044] A reported.sup.[21] one-pot method for synthesis of
.alpha.-nitro acids was investigated which involved the use of
magnesium methyl carbonate (MMC) as a carboxylating agent to
introduce a carboxyl group at the .alpha.-carbon of a primary
nitroalkane (Scheme 5): 17
[0045] When 1-nitropropane was used as the starting material, the
.sup.1H NMR of the residue obtained after workup indicated
formation of the corresponding .alpha.-nitro carboxylic acid
However, when the long chain nitroalkane 4a was used as the
starting material, the expected .alpha.-nitro acid product 13a was
not detected in the crude reaction mixture. The lack of reaction
for stearyl nitrite may be attributed to poor solubility of stearyl
nitrite in MMC solution.
[0046] Synthesis of the .alpha.-nitro acids 14a was subsequently
investigated by conversion of the nitroalkane 4a of the
corresponding .alpha.-nitro acid ester 14a by treatment with methyl
chloroformate, followed by hydrolysis (Scheme 6): 18
[0047] Using this scheme, the saturated nitro acid ester 13a was
obtained in 25% yield from the corresponding nitroalkane 4a.
Treatment of the ester 14a with lithium hydroxide in
dimethoxyethane (DME) did not give rise to the desired add 13a. The
nitroalkane 4a, however, was isolated as the sole product of this
reaction. This result can be explained as illustrated in Scheme 7.
19
[0048] It has been reported.sup.[22] that free .alpha.-nitroacetic
acid and its dianion salt are quite stable at room temperature, but
that the monoanion salt decarboxylates rapidly at room temperature.
The failure in generating the .alpha.-nitropropanoic acid is then
likely due to decarboxylation of the monoanion in the basic
reaction medium.
[0049] (6) Synthesis of Hydroxy and Hydroperoxy Derivatives of
Compound 6b
[0050] Synthesis of hydroxy and hydroperoxy products of compound 6b
was based on Scheme 8. Pure compound 17 was obtained in the yield
of 32%. Compound 16 was relatively unstable, but the product with
90% purity was obtained by column chromatography at 0.degree. C.,
and was used for investigation of its inhibitory effect on 15-LO
catalysed oxidation of arachidonic acid. 20
[0051] (7) Synthesis of Polyunsaturated Nitroalkanes and
Nitro-Substituted Fatty Acids.
[0052] The polyunsaturated fatty alcohols 1b-e and the saturated
analogue, octadecanol 1a, are commercially available and were used
as starting materials. Their treatment with triphenylphosphine and
carbon tetrabromide according to the method of Hayashi et
al..sup.(23) afforded the corresponding bromides 2a-e. Short chain
bromoalkanes react with silver nitrite to give
nitroalkanes.sup.(24) but the bromides 2a-e were inert to such
treatment. Instead, they were first treated with sodium iodide to
give the iodides 3a-e, which were used without purification and
converted to the nitroalkanes 4a-e, respectively. 21
[0053] In order to prepare nitro-substituted fatty acids, a variety
of reactions of nitroalkanes were investigated. Carboxylation using
the method of Finkbeiner et al..sup.(25) was examined. Accordingly
reaction of 1-nitropropane with magnesium methyl carbonate afforded
2-nitrobutanoic acid, but 1-nitrooctadecane (4a) was recovered
unchanged when treated under the same conditions. Apparently the
aliphatic chain prevents reaction in the latter case.
1-Nitrooctadecane (4a) was treated with butyl lithium then methyl
chloroformate.sup.(26) to give methyl 2-nitrononadecanoate.
However, all attempts to hydrolyse this material to give
2-nitrononadecanoic acid failed, the reactions instead affording
the nitroalkane 4a. 22
[0054] This product (ie nitroalkane 4a) may be attributed to rapid
decarboxylation of the monoanion of 2-nitrononadecanoic acid, since
the analogous process has been reported for 2-nitroacetic
acid..sup.(27) Given that this decarboxylation would be expected to
affect the integrity of 2-nitrocarboxylic acids during
physiological studies at near neutral pH, the synthesis of
compounds of this type was not further pursued.
[0055] The nitroalkane 4a was inert when treated with butyl lithium
and .alpha.-haloacetates, indicating that long chain
3-nitrocarboxylates could not be prepared using this approach.
However, the nitroalkanes 4a,b reacted with sodium hydroxide and
methyl acrylate.sup.(28) in the presence of tetrabutylammonium
iodide.sup.(29) to give the .gamma.-nitroesters 5a,b, which were
hydrolysed using lithium hydroxide to afford the corresponding
nitroacids 6a,b. Using 1,8diazobicyclo[5.4.0]undec-7-ene (DBU) as
the base, in place of sodium hydroxide, the nitroalkanes 4a,b
reacted by sequential Michael additions with methyl acrylate to
give the diesters 7a,b, which hydrolysed to the nitrodiacids 8a,b.
23
[0056] To obtain substituted nitroalkanes, the alcohols 1a,b were
oxidised to the corresponding aldehydes 9a,b using pyridinium
chlorochromate..sup.(30) Henry condensation.sup.(31) of these
compounds with nitromethane in the presence of Amberlyst
A-21.sup.(32) afforded the 2-hydroxynitroalkanes 10a,b, which
reacted with methanesulfonyl chloride and triethylamine.sup.(33) to
give the corresponding .alpha.,.beta.-unsaturated nitroalkanes.
Unfortunately it was not possible to isolate pure samples of these
analogues of .alpha.,.beta.-unsaturated fatty acids, because they
equilibrated with the corresponding .beta.,.gamma.-unsaturated
nitroalkanes and the mixtures of isomers decomposed on
chromatography. 24 25
[0057] The reactions described above were carried out under
nitrogen and in the dark. After purification the compounds were
stored at -30.degree. C. under nitrogen. By taking these
precautions there were no complications from isomerisation or
autoxidation of the methylene-interrupted polyenes. Such reactions
result in the formation of conjugated dienes and none of the
compounds showed absorption at 234 nm which is characteristic of
this structural feature..sup.(34)
[0058] Experimental
[0059] Octadecan-1-ol (1a) was obtained from Aldrich Chemical Co.
Arachidonyl alcohol (1b), linolenyl alcohol (1c), gamma linolenyl
alcohol (1d) and docosahexaenyl alcohol 1e were purchased from
Nu-Chek Prep. Inc. (Elysian, Minn., USA).
[0060] 1-Bromooctadecane (2a); Typical Procedure
[0061] Octadecan-1-ol (1a) (520 mg, 1.92 mmol) and Ph.sub.3P (530
mg, 2.10 mmol) were dissolved in CH.sub.2Cl.sub.2 (25 mL). The
mixture was cooled in an ice bath and CBr.sub.4 (630 mg, 1.90 mmol)
was added with stirring. The mixture was allowed to warm to r.t.
and was stirred overnight, then it was concentrated under a stream
of N.sub.2 and the residue was subjected to flash column
chromatography on silica, eluting with hexane, to afford
1-bromooctadecane (2a) (605 mg, 96%) as a waxy solid; mp
26-28.degree. C.
[0062] IR (KBr): .nu.=2920 (s), 2848 (s), 1468 (s), 1378 (w), 1254
(w), 1144 (m), 720 (m), 658 (s) cm-1
[0063] .sup.1H NMR (CDCl.sub.3, 300 MHz): .delta.=0.87 (t, 3H,
J=6.7 Hz, C18-H.sub.3), 1.25-1.32 [m, 30H, (C3-17)-H.sub.2)],
1.82-1.85 (m, 2H, C2-H.sub.2), 3.40 (t, 2H, J=6.8 Hz,
C1-H.sub.2).
[0064] .sup.13C NMR (CDCl.sub.3, 300 MHz): .delta.=14.7, 23.2,
28.7, 29.3, 29.9, 30.0, 30.1, 30.1(6), 30.2(3), 32.5, 33.4,
34.6.
[0065] MS (EI): m/z (%)=334 (M.sup.+, 8), 332 (M.sup.+, 10), 253
(25), 131 (27), 149 (28), 137 (67), 135 (69), 113 (19), 97 (30), 85
(30), 71 (70), 57 (100).
[0066] HRMS: m/z calcd for C.sub.13H.sub.37Br 334.2058 (M.sup.+)
and 332.2078 (M.sup.+). Found: 334.2070 and 332.2086.
[0067] (all-Z)-1-Bromo-5,8,11,14-eicosatetraene (2b)
[0068] From arachidonyl alcohol (1b) (740 mg, 2.54 mmol), using the
procedure described above for preparation of 1-bromooctadecane
(2a), (all-Z)-1-bromo-5,8,11,14-eicosatetraene (2b) (826 mg, 93%)
was obtained as a colourless oil.
[0069] IR (film): .nu.=3012 (s), 2958 (s), 2927 (s), 2856 (s), 1653
(m), 1456 (m), 1394 (m), 1251 (m), 1199 (w), 1041 (m), 913 (w), 807
(w), 715 (s) cm.sup.-1.
[0070] .sup.1H NMR (CDCl.sub.3, 300 MHz): .delta.=0.89 (t, 3H,
1=6.8 Hz, C203), 1.29-1.38 (m, 6H, C17-H2, C18-H2, C19-H2);
1.47-1.56 (m, 2H, C3-H.sub.2), 1.83-1.93 (m, 2H, C2-H2), 2.03-214
(m, 4H, C4 Hz, C16-H2), 2.80-2.83 (m, 6H, C7-Hz C10-H2,
C13-H.sub.2), 3.42 (t, 2H, J=6.8 Hz, C1-H.sub.2), 5.30-5.41 (m, 8H,
CG-H, C6H, C8-H, C9-H, C11-H, C12-H, C14-H, C15-H).
[0071] .sup.13C NMR (CDCl.sub.3, 300 MHz): .delta.=14.7, 23.2,
26.2, 26.9, 27.8, 28.7, 29.9, 32.1, 32.9, 34.3, 128.1, 128.4, 128.7
(2C), 129.0, 129.1, 129.9, 131.1.
[0072] MS (EI): m/z (%)=354 (M.sup.+, 5), 352 (M.sup.+, 6), 283
(8), 281 (8), 256 (15), 254 (15), 216 (25), 214 (25), 150 (34), 119
(29), 105 (36), 93 (53), 91 (56), 79 (100), 67 (75).
[0073] HRMS: m/z calcd for C.sub.20H.sub.33Br 354.1745 (M.sup.+)
and 352.1766 (M.sup.+). Found: 354.1748 and 352.1772.
[0074] Anal. Calcd for C.sub.20H.sub.33Br: C, 67.98; H, 9.41.
Found: C, 68.05; H, 9.28.
[0075] (Z,Z,Z)-1-Bromo-9,12,13-octadecatriene (2c)
[0076] From linolenyl alcohol (1c) (102 mg, 0.39 mmol), using the
procedure described above for preparation of 1-bromooctadecane
(2a), (Z,Z,Z)-1-bromo-9,12,15-octadecatriene (2c) (118 mg, 93%) was
obtained as a colourless oil.
[0077] IR (film): .nu.=3001 (s), 2960 (s), 2920 (s), 2850 (s), 1460
(m), 1430 (m), 1395 (w), 1270 (w), 720 (w) cm.sup.-1.
[0078] .sup.1H NMR (CDCl.sub.3, 300 MHz): .delta.=0.98 (t, 3H,
J=7.5 Hz, C18-H3), 1.30-1.45 (m, 10H, C3-H.sub.2, C.sub.4H.sub.2,
C5-H.sub.2, C6-H.sub.2, C7-H.sub.2), 1.81-1.88 (m, 2H, C2-H.sub.2),
2.03-2.11 (m, 4H, C8-H, C17-H.sub.2), 2.80-2.83 (m, 4H,
C11-H.sub.2, C14-H.sub.2), 3.41 (t, 2H, J=6.8 Hz, C1-H.sub.2),
5.30-5.42 (m, 6H, C9-H, C10-H, C12-H, C13-H, C15-H, C16H).
[0079] .sup.13C NMR (CDCl.sub.3, 300 MHz): .delta.=14.9, 21.1,
26.1, 26.2, 27.8, 28.7, 29.3, 29.8, 29.9, 30.2, 33.4, 34.6, 127.7,
128.3, 128.8 (2C), 130.8, 132.5.
[0080] MS (EI): m/z (%)=328 (M.sup.+, 14), 326 (M.sup.+, 14), 272
(42), 270 (41), 149 (13), 135 (28), 121 (33), 108 (92), 95 (53), 79
(100), 67 (72), 55 (59).
[0081] Anal. Calcd for C.sub.18H.sub.31Br: C, 66.05; H, 9.54.
Found: C, 65.82; H, 9.32.
[0082] (Z,Z,Z)-1-Bromo-6,9,12-octadecatriene (2d)
[0083] From gamma linolenyl alcohol (1d) (143 mg, 0.54 mmol), using
the procedure described above for preparation of 1-bromooctadecane
(2a), (Z,Z,Z)-1-bromo-6,9,12-octadeca-triene (2d) (170 mg, 96%) was
obtained as a colourless oil.
[0084] IR (film): .nu.=3002 (s), 2950 (s), 2920 (s), 2850 (s), 1460
(s), 1378 (w), 1260 (w), 713 (m), 648 (m) cm.sup.-1.
[0085] .sup.1H NMR (CDCl.sub.3, 300 MHz): .delta.=0.89 (t, 3H,
J=6.8 Hz, C18-H3), 1.29-1.43 (m, 10H, C3-Hz, C4-Hz, C13-H.sub.2,
C16-Hz, C17-Hz), 1.82-1.91 (m, 2H, C2-H.sub.2), 2.02-2.17 (m, 4H,
C5-Hz C14-H.sub.2), 2.79-2.83 (m, 4H, C8-H.sub.2, C11-H.sub.2),
3.40 (t, 2H, J=6.7 Hz, C1-H.sub.2), 5.30-5.41 (m, 6H, C6-H, C7-H,
C9-H, C10-H, C12-H, C13-H).
[0086] .sup.13C NMR (CDCl.sub.3, 300 MHz): .delta.=14.7, 23.2,
26.2, 27.6, 27.8, 28.4, 29.3, 29.9, 32.1, 33.3, 34.5, 128.2,
128.6(5), 128.7(1), 129.0, 130.3, 131.0.
[0087] MS (EI): m/z (%)=328 (M.sup.+, 10), 326 (M.sup.+, 8), 230
(49), 228 (50), 150 (66), 135 (15), 121 (25), 107 (32), 93 (59), 79
(100), 67 (95), 55 (64).
[0088] HRMS: m/z calcd for C.sub.15H.sub.31Br 328.1589 (M.sup.+)
and 326.1609 (M.sup.+). Found: 328.1592 and 326.1611.
[0089] (all-Z)-1-Bromo-4,7,10,13,16,19-docosahexaene (2e)
[0090] From docosahexaenyl alcohol 1e (201 mg, 0.64 mmol), using
the procedure described above for preparation of 1-bromooctadecane
(2a), (all-Z)-1-bromo-4,7,10,13,16,19-docosahexaene (2e) (221 mg,
92%) was obtained as a colourless oil.
[0091] IR (film): .nu.=3008 (s), 2960 (s), 2928 (s), 2868 (s), 1650
(m), 1434 (s), 1392 (s), 1348 (w), 1322 (w), 1266 (s), 1244 (s),
1068 (m), 1044 (m), 928 (m), 714 (s) cm.sup.-1.
[0092] .sup.1H NMR (CDCl.sub.3, 300 MHz): .delta.=0.98 (t, 3H,
J=7.5 Hz, C22-H3), 1.85-2.30 (6H, C2-H.sub.2, C3-H.sub.2, C21-H2),
2.80-2.90 (m, 10H, C6-Hz C9-H.sub.2, C12-H2, C15-Hz, C18-H.sub.2),
3.42 (t, 2H, J=6.6 Hz, C1-H2), 5.31-5.45 (m, 12H, C4-H, C5-H, C7-H,
C8-H, C10-H, C11-H, C13-H, C14-H, C16H, C17-H, C19-H, C20-H).
[0093] .sup.13C NMR (CDCl.sub.3, 300 MHz): S=14.4, 20.5, 25.5,
25.6, 32.5 33.2, 127.0, 127.8(5), 127.9(4), 128.0(6), 128.1(1)
(2C), 128.1(8) (2C), 1281(4), 128.6, 129.5, 132.0.
[0094] MS (EI): m/z (%)=378 (M.sup.+, 10), 376 (M.sup.+, 10), 349
(20), 347 (20), 309 (46), 307 (53), 244 (75), 242 (74), 227 (49),
202 (30), 200 (30), 173 (12), 133 (34), 119 (45), 108 (50), 91
(65), 79 (100), 67 (66).
[0095] HRMS: m/z calcd for C.sub.22H.sub.33Br 378.1745 (M.sup.+)
and 376.1766 (M.sup.+). Found: 378.1742 and 376.1760.
[0096] 1-Nitrooctadecane (4a); Typical Procedure
[0097] To a solution of 1-bromooctadecane (2a) (480 mg, 1.44 mmol)
in dry acetone (25 mL) at r.t. was added NaI (430 mg, 2.87 mmol).
The mixture was stirred at r.t. overnight, then the solvent was
removed in vacuo. The residue was mixed with 25 mL of sat. aq
sodium bisulfite and the mixture was extracted with Et.sub.2O
(3.times.25 mL). The combined extracts were dried
(Na.sub.2SO.sub.4) and the solvent was removed in vacuo. The
residue (502 mg) was dissolved in anhyd Et.sub.2O and AgNO.sub.2
(406 mg, 2.64 mmol) was added. After 3 days of stirring, the
mixture was filtered through a bed of celite and the filtrate was
evaporated under a stream of dry N.sub.2. The residue was subjected
to flash column chromatography on silica (Et.sub.2O/hexane, 5/95)
to give crude iodide 3a (97 mg) and 1-nitrooctadecane (4a) (220 mg,
51%) as a white solid; mp 4142.degree. C.
[0098] IR (film): .nu.=2954 (s), 2919 (s), 2850 (s), 1563 (s), 1470
(m), 1385 (w), 1147 (w), 742 (w), 720 (m), 650 (w) cm.sup.-1.
[0099] .sup.1H NMR (CDCl.sub.3, 300 MHz): .delta.=0.88 (t, 3H,
J=6.6 Hz, C18-H.sub.3), 1.25-1.34 [m, 30H, (C3-C17)-H.sub.2],
1.96-2.05 (m, 2H, C2-H.sub.2), 4.38 (t, 2H, J=7.1 Hz,
C1-H.sub.2).
[0100] .sup.13C NMR (CDCl.sub.3, 300 MHz): .delta.=14.7, 23.3,
26.7, 28.0, 29.4, 29.8, 29.9, 30.0, 30.1, 30.2, 30.3, 32.5,
76.3.
[0101] MS (EI): m/z (%) 299 (M.sup.+, <1), 282 (4), 264 (20),
252 (7), 238 (7), 224 (7), 210 (5), 196 (4), 134 (J), 139 (7), 125
(20), 111 (40), 97 (74), 83 (87), 69 (95), 57 (100), 55 (96).
[0102] Anal. Calcd for C.sub.18H.sub.37NO.sub.2: C, 72.19; H,
12.45; N, 4.68. Found: C, 72.33; H, 12.77; N, 4.57.
[0103] (all-Z)-1-Nitro-5,8,11,14-eicosatetraene (4b)
[0104] According to the procedure described above for preparation
of 1-nitrooctadecane (4a), (all-Z)-1-bromo-5,8,11,14-eicosatetraene
(2b) (782 mg, 2.21 mmol) gave crude iodide (3b) (71 mg) and
(all-Z)-1-nitro-5,8,11,14-eicosatetraene (4b) (397 mg, 56%) as a
colourless oil.
[0105] IR (film): .nu.=3013 (s), 2957 (s), 2928 (s), 2857 (s), 1648
(w), 1555 (s), 1457 (m), 1433 (m), 1381 (s), 1267 (w), 1106 (w),
1047 (w), 969 (w), 914 (w), 716 (m) cm.sup.-1.
[0106] .sup.1H NMR (CDCl.sub.3, 300 MHz): .delta.=0.89 (t, 3H,
J=6.8 Hz, C20-H3), 1.20-1.51 (m, 8H, C3-C.sub.2, C17-H.sub.2,
C18-H.sub.2, C19-H.sub.2), 1.99-2.16 (m, 6H, C2-H.sub.2,
C.sub.4H.sub.2, C16-H.sub.2), 2.79-2.86 (m, 6H, C7-H, C10-H2,
C13-H2), 4.39 (t, 2H, J=7.0 Hz, C1-H.sub.2), 5.32-5.43 (m, 8H,
C5-H, C6-H, C8-H, C9-H, C11-H, C12-H, C14-H, C5-H).
[0107] .sup.13C NMR (CDCl.sub.3, 300 MHz): .delta.=14.6, 23.1,
26.2, 26.7, 26.9, 27.5, 27.8 29.9, 32.1, 76.1, 128.1, 128.4, 128.5,
128.9, 129.2 (2C), 129.6, 131.1.
[0108] MS (EI): m/z (%)=319 (M.sup.+, 6), 302 (14), 220 (27), 205
(15), 190 (11), 181 (24), 177 (20), 164 (25), 150 (41), 119 (48),
105 (63), 91 (90), 79 (100), 67 (97), 55 (77).
[0109] Anal. Calcd for C.sub.20H.sub.33NO.sub.2: C, 75.19; H,
10.41; N, 4.38. Found: C, 74.92; H, 10.40; N, 4.43.
[0110] (Z,Z,Z)-1-Nitro-9,12,15-octadecatriene (4c)
[0111] Following the procedure described above for preparation of
1-nitrooctadecane (4a), (Z,Z,Z)-1-bromo-9,12,15-octadecatriene (2c)
(79 mg 0.24 mmol) gave crude iodide 3c (12 mg) and
(Z,Z,Z)-1-nitro-9,12,15-oc- tadecatriene (4c) (37 mg, 53%) as a
colourless oil.
[0112] IR (film): .nu.=3011 (s), 2962 (s), 2929 (s), 2856 (s), 1652
(w), 1554 (s), 1463 (m), 1433 (m), 1383 (m), 1268 (w), 1148 (w),
1069 (w), 968 (m), 912 (w), 724 (m), 614 (w) cm.sup.-1.
[0113] .sup.1H NMR (CDCl.sub.3, 300 MHz): .delta.=0.98 (t, 3H,
J=7.5 Hz, C18-H.sub.3), 1.25-1.33 (m, 10H, C3-H.sub.2, C4-H.sub.2,
C5-H.sub.2, C6-H.sub.2, C7-H.sub.2), 1.97-2.06 (m, 6H, C2-H.sub.2,
C8-H.sub.2, C17-H.sub.2), 2.79-2.81 (m, 4H, C11-H.sub.2,
C14-H.sub.2), 4.37 (t, 2H, J=7.1 Hz, C1-H.sub.2), 5.36-5.40 (m, 6H,
C9-H, C10-H, C12-H, C13-H, C15-H, C16-H).
[0114] .sup.13C NMR (CDCl.sub.3, 300 MHz): .delta.=14.9, 21.1,
26.1, 26.2, 26.8, 27.7, 28.0, 29.4, 29.6, 29.7, 30.1, 76.3, 127.7,
128.4, 128.8, 128.9, 130.7, 132.5.
[0115] MS (EI): m/z (%)=293 (M.sup.+, 24), 276 (14), 264 (5), 246
(5), 237 (32), 224 (17), 135 (26), 121 (33), 108 (63), 95 (84), 93
(75), 91 (69), 79 (100), 67 (95).
[0116] Anal. Calcd for C.sub.18H.sub.31NO.sub.2: C, 73.67; H,
10.65; N, 4.77. Found: C, 73.69; H, 10.57; N, 4.85.
[0117] (Z,Z,Z)-1-Nitro-6,9,12-octadecatriene (4d)
[0118] Following the procedure described above for preparation of
1-nitrooctadecane (4a), (Z,Z,Z)-1-bromo-6,9,12-octadecatriene (2d)
(122 mg, 0.37 mmol) gave crude iodide 3d (13 mg) and
(Z,Z,Z)-1-nitro-6,9,12-oc- tadecatriene (4d) (56 mg, 51%) as a
colourless oil.
[0119] IR (film): .nu.=3012 (s), 2956 (s), 2928 (s), 2858 (s), 1652
(m), 1555 (s), 1464 (s), 1433 (s), 1382 (s), 1266 (m), 1159 (w),
1067 (w), 1040 (w), 970 (w), 914 (w), 720 (s), 614 (w)
cm.sup.-1.
[0120] .sup.1H NMR (CDCl.sub.3, 300 MHz): .delta.=0.88 (t, 3H,
J=7.1 Hz, C18-H), 1.29-1.43 (m, 10H, C3-H1, C4-H.sub.2, C15-H1,
C16-H.sub.2, C17-H.sub.2), 2.01-2.08 (m, 6H, C2-Hz, C5-H.sub.2,
C14-H.sub.2), 2.78-2.82 (m, 4H, C8-H, C011-H.sub.2), 4.38 (t, 2H,
J=7.1 Hz, C1-H2), 5.34-5.40 (m, 6H, C6-H, C7-H, C9-H, C10-H, C12-H,
C13-H).
[0121] .sup.13C NMR (CDCl.sub.3, 300 MHz): .delta.=14.7, 23.2,
26.2, 26.4, 27.4, 27.8, 27.9, 29.4, 29.9, 32.1, 76.2, 128.1, 128.5,
129.0 (2C), 129.9, 131.0.
[0122] MS (EI): m/z (%)=293 (M.sup.+, 31), 276 (25), 258 (12), 246
(4), 222 (7), 195 (72), 150 (36), 137 (18), 105 (25), 91 (84), 81
(80), 80 (79), 79 (100), 67 (82), 55 (60).
[0123] Anal. Calcd for C.sub.18H.sub.31NO.sub.2: C, 73.67; H,
10.65; N, 4.77. Found: C, 73.56; H, 10.56; N, 4.74.
[0124] (all-Z)-1-Nitro-4,7,10,13,16,19-docosahexaene (4e)
[0125] Following the procedure described above for preparation of
1-nitrooctadecane (4a),
(all-Z)-1-bromo-4,7,10,13,16,19-docosahexaene (2e) (165 mg, 0.44
mmol) gave crude iodide 3e (27 mg) and
(all-Z)-1-nitro-4,7,10,13,16,19-docosahexaene (4e) (80 mg, 53%) as
a colourless oil.
[0126] IR (film): .nu.=3014 (s), 2962 (s), 2926 (s), 2873 (s), 2854
(s), 1653 (m), 1554 (s), 1434 (s), 1381 (s), 1352 (m), 1267 (m),
1069 (w), 917 (w), 712 (s), 611 (w) cm.sup.-1.
[0127] .sup.1H NMR (CDCl.sub.3, 300-MHz): .delta.=0.98 (t, 3H,
J=7.6 Hz, C22-H.sub.3), 2.05-2.23 (m, 6H, C2-H2, C3-Hz
C21-H.sub.2), 2.78-2.85 (m, 10H, C6-H.sub.2, C9-H.sub.2, C12-H2,
C15-H.sub.2, C18-H.sub.2), 4.38 (t, 2H, J=6.7 Hz, C1-H.sub.2),
5.31-5.47 (m, 12H, C4-H, C5-H, C7-H, C8-H, C10H, C11-H, C13-H,
C14-H, C16-H, C17-H, C19-H, C20-H).
[0128] .sup.13C NMR (CDCl.sub.3, 300 MHz): .delta.=14.8, 21.1,
24.4, 26.1, 26.2, 27.7, 75.4, 127.6, 128.3, 128.4, 128.5(5),
128.6(0), 128.9 (3C), 129.1, 129.2, 130.9, 132.6.
[0129] MS (EI): m/z (%)=343 (M.sup.+, 10), 326 (59), 314 (21), 274
(44), 215 (55), 207 (42), 167 (16), 145 (18), 131 (16), 119 (36),
105 (48), 91 (77), 79 (100), 67 (78), 53 (42).
[0130] Anal. Calcd for C.sub.22H.sub.33NO.sub.2: C, 76.92; H, 9.68;
N, 4.08. Found: C, 76.52; H, 9.87; N, 4.26.
[0131] Methyl 4-Nitroheneicosanoate (5a); Typical Procedure
[0132] A solution of NaOH (136 mg, 3.4 mmol) and Bu.sub.4NI (158
mg, 0.43 mmol) in water (10 mL) was added to a solution of
1-nitrooctadecane (4a) (510 mg, 1.70 mmol) and methyl acrylate (442
mg, 5.13 mmol) in CH.sub.2Cl.sub.2 (10 mL) at r.t. The mixture was
stirred and heated at reflux for 24 h, then it was cooled and the
layers were separated. The organic phase was washed with water
(2.times.25 mL) and dried with Na.sub.2SO.sub.4. The solvent was
evaporated and the residue was subjected to flash column
chromatography on silica (Et.sub.2O/hexane, 5/95), giving methyl
4-nitroheneicosanoate (5a) (498 mg, 76%) as a waxy solid.
[0133] IR (Nujol): .nu.=2924 (s), 2853 (s), 1744 (s), 1554 (s),
1466 (m), 1439 (m), 1367 (m), 1201 (m), 1175 (m), 1120 (m), 829
(w), 722 (w) cm.sup.1.
[0134] .sup.1H NMR (CDCl.sub.3, 300 MHz): .delta. 0.87 (t, 3H,
J=6.7 Hz, C21-H3), 1.19-1.25 [m, 30H, (C6-C20)-Hz], 1.69-1.78 (m,
1H), 1.92-2.30 (m, 3H), 2.32-2.40 (m, 2H, C2-H2), 3.69 (s, 3H,
OCH.sub.3), 4.50-4.59 (m, 1H, C4-H).
[0135] .sup.13C NMR (CDCl.sub.3, 300 MHz): .delta.=14.7, 23.3,
26.2, 29.2, 29.5, 29.8, 29.9, 30.0, 30.1, 302, 30.3, 30.5, 32.5,
34.5, 52.5, 88.4, 173.0.
[0136] MS (EI): m/z (%)=386 [(M+1).sup.+, 251, 368 (12), 354 (18),
339 (20), 305 (24), 287 (28), 263 (18), 221 (15), 193 (10), 179
(15), 165 (21), 151 (26), 137 (31), 123 (36), 111 (52), 97 (76), 83
(86), 69 (88), 55 (100).
[0137] HRMS: m/z calcd for C.sub.22H.sub.44NO.sub.4 386.3270
(M+H).sup.+. Found 386.3275.
[0138] Anal. Calcd for C.sub.22H.sub.43NO.sub.4: C, 68.53; H,
11.24; N, 3.63. Found: C, 68.39; H, 11.53; N, 3.50.
[0139] Methyl (all-Z)-4-Nitrotricosa-8,11,14,17-tetraenoate
(5b)
[0140] Following the procedure described above for preparation of
methyl 4-nitroheneicosanoate (5a),
(all-Z)-1-nitro-5,8,11,14-eicosatetraene (4b) (650 mg, 2.03 mmol)
gave methyl (all-Z)-4-nitrotricosa-8,11,14,17-tetraen- oate (5b)
(594 mg, 72% O) as a colourless oil.
[0141] IR (film): .nu.=3065 (w), 3013 (m), 2956 (s), 2930 (s), 2859
(m), 1737(s), 1552 (s), 1439 (m), 1363 (w), 1267 (w), 1263 (w),
1259 (w), 1204 (m), 1178 (m), 981 (w) cm.sup.-1.
[0142] .sup.1H NMR (CDCl.sub.3, 300 MHz): .delta.=0.88 (t, 3H,
J=6.8 Hz, C23-H3), 1.24-1.45 (m, 8H, C6-H.sub.2, C.sub.20H.sub.2,
C21-H.sub.2, C22-H.sub.2), 1.70-1.81 (m, 1H), 1.91-2.27 (m, 7H),
2.32-2.40 (m, 2H, C2-H.sub.2), 2.73-2.83 (m, 6H, C10-H.sub.2,
C13-H.sub.2, C16-H.sub.2), 3.68 (s, 3H, OCH.sub.3), 4.514.58 (m,
1H, C4-H), 5.29-5.44 (m, 8H, C8-H, C9-H, C11-H, C12-H, C14-H,
C15-H, C17-H, C18-H).
[0143] .sup.13C NMR (CDCl.sub.3, 300 MHz): .delta.=14.7, 23.1,
26.1, 26.2, 26.9, 27.8, 29.2, 29.9, 30.3, 30.5, 32.1, 33.9, 52.5,
88.2, 128.1, 128.4, 128.6, 128.9, 129.2 (2C), 129.6, 131.1,
172.9.
[0144] MS (EI): m/z (%)=405 (NI+, 7), 374 (8), 359 (5), 327 (4),
307 (15), 294 (6), 267 (4), 229 (5), 215 (10), 190 (13), 177 (27),
164 (33), 150 (36), 147 (24), 131 (35), 119 (43), 105 (54), 91
(70), 79 (93), 67 (100), 55 (56).
[0145] HRMS: m/z calcd for C.sub.24H.sub.39NO.sub.4 405.2879
(M.sup.+). Found 405.2870.
[0146] Anal. Calcd for C.sub.24H.sub.39NO.sub.4: C, 71.08; H, 9.69;
N, 3.45. Found: C, 71.50; H, 10.03; N, 3.34.
[0147] 4-Nitroheneicosanoic Acid (6a); Typical Procedure
[0148] Methyl 4-nitroheneicosanoate (5a) (147 mg, 0.38 mmol) was
dissolved in 1,2-dimethoxyethane (DME) (2 mL) and sat. aq LiOH
solution (2 mL) was added. The mixture was left for 24 h, then it
was acidified with dilute HCl (10%, 10 mL) and the mixture was
extracted with EtOAc (2.times.10 mL). The extracts were
concentrated under a stream of dry N.sub.2 and the residue was
subjected to flash column chromatography on silica
(Et.sub.2O/hexane, 100/20, then Et.sub.2O/hexane/HOAc, 60/40/1) to
afford 4-nitroheneicosanoic acid (6a) (121 mg, 85%) as a white
solid; mp 55-56.degree. C.
[0149] IR (KBr): .nu.=3500-2600 (br), 2955 (m), 2919 (s), 2849 (s),
1698 (s), 1615 (w), 1543 (s), 1467 (m), 1445 (m), 1413 (w), 1360
(w), 1334 (w), 1266 (w), 923 (w), 827 (w), 723 (w), 612 (w)
cm.sup.-1.
[0150] .sup.1H NMR (CDCl.sub.3, 300 MHz): .delta.=0.87 (t, 3H,
J=7.1 Hz, C21-H3), 1.20-1.28 [m, 30H, (C6-C20)-H.sub.2], 1.69-1.78
(m, 1H), 1.98-2.30 (m, 3H), 2.39-2.48 (m, 2H, C2-H.sub.2),
4.53-4.60 (m, 1H, C4-H).
[0151] .sup.13C NMR (CDCl.sub.3, 300 MHz): .delta.=14.7, 23.3,
26.2, 28.8, 29.5, 29.8, 29.9, 30.0, 30.1, 30.2(6), 30.3(3), 32.5,
34.4, 88.2, 177.5.
[0152] MS (CI): m/z=389.3 (M+NH.sub.4).sup.+.
[0153] MS (EI): m/z (%)=354 [(M-OH).sup.+, 2], 323 (19), 321 (19),
305 (17), 287 (14), 263 (12), 236 (5), 221 (9), 193 (10), 179 (15),
165 (15), 151 (17), 137 (20), 125 (25), 110 (73), 97 (100), 83
(64), 69 (64), 55 (73).
[0154] HRMS: m/z calcd for C.sub.21H.sub.40NO.sub.3 354.3008
(M-OH).sup.+. Found 354.3006.
[0155] Anal. Calcd for C.sub.21H.sub.41NO.sub.4: C, 67.88; H,
11.12; N, 3.77. Found: C, 67.58; H, 11.08; N, 3.81.
[0156] (all-Z).sub.4-Nitrotricosa-8,11,14,17-tetraenoic Acid
(6b)
[0157] Following the procedure described above for preparation of
4-nitroheneicosanoic acid (6a), methyl
(all-Z)-4-nitrotricosa-8,11,14,17-- tetraenoate (5b) (230 mg, 0.57
mmol) gave (all-Z)-4-nitrotricosa-8,11,14,1- 7-tetraenoic acid (6b)
(207 mg, 93%) as a colourless oil.
[0158] IR (film): .nu.=3611-3317 (br), 3013 (m), 2922 (s), 2852
(m), 2693 (m), 2361 (w), 1714 (s), 1551 (s), 1441 (s), 1379 (m),
1360 (m), 1270 (m), 1071 (m), 969 (w), 916 (m), 844 (m), 824 (w),
720 (m) cm.sup.-1.
[0159] .sup.1H NMR (CDCl.sub.3, 300 MHz): .delta.=0.89 (t, 3H,
J=7.1 Hz, C23-H.sub.2), 1.27-1.44 (m, 8H, C6-H.sub.2, C20-H.sub.2,
C21-H2, C22-H.sub.2), 1.70-1.82 (m, 1H), 1.93-2.27 (m, 7H),
240-2.48 (m, 2H, C2-H.sub.2), 2.78-2.86 (m, 6H, C10-H2, C13-H2,
C16-H2), 4.564.59 (m, 1H, C4-H), 5.305.43 (m, 8H, C8-H, C9-H,
C11-H, C12-H, C14-H, C15-H, C17-H, C18-H).
[0160] .sup.13C NMR (CDCl.sub.3, 300 MHz): .delta.=14.7, 23.1,
26.1, 26.2, 26.9, 27.8, 28.9, 29.9, 30.2, 32.1, 33.9, 88.1, 128.1,
128.4, 128.5, 128.9, 129.1, 129.2, 129.7, 131.1, 176.8.
[0161] MS (EI): m/z (%) 391 (M.sup.+, 8), 343 (8), 320 (4), 293
(13), 280 (8), 253 (10), 203 (15), 190 (25), 177 (28), 164 (42),
150 (46), 131 (34), 110 (100), 91 (72), 79 (93), 67 (97).
[0162] HRMS: m/z calcd for C.sub.23H.sub.37NO.sub.4 391.2723
(M.sup.+). Found 391.2725.
[0163] Anal. Calcd for C.sub.3H.sub.37NO.sub.4: C, 70.55; H, 9.52;
N, 3.58. Found: C, 70.29; H, 9.86; N, 3.43.
[0164] Dimethyl 3-Heptadecyl-3-nitropentane-1,5-dicarboxylate (7a);
Typical Procedure
[0165] A solution containing 1-nitrooctadecane (4a) (50 mg, 0.17
mmol), methyl acrylate (88 mg, 1.02 mmol) and DBU (13 mg, 0.085
mmol) in CH.sub.2Cl.sub.2 (2 mL) was kept at r.t. for 24 h, then it
was acidified with HCl (10%, 5 mL) and the mixture was extracted
with CH.sub.2Cl.sub.2 (2.times.10 mL). The combined extracts were
dried with Na.sub.2SO.sub.4 and concentrated, and the residue was
subjected to flash column chromatography on silica (EtOAc/petroleum
spirit, 15/85), to give dimethyl
3-heptadecyl-3-nitropentane-1,5-dicarboxylate (7a) (76 mg, 95%) as
a colourless oil.
[0166] IR (film): .nu.=2954 (m), 2914 (s), 2849 (s), 1744 (s), 1732
(s), 1537 (s), 1470 (s), 1458 (s) 1439 (s), 1378 (s), 1355 (s),
1319 (s), 1298 (s), 1203 (s), 1180 (s), 1129 (s), 1110 (m), 1071
(m), 1022 (m), 986 (s), 894 (s), 864 (m), 842 (s), 826 (s), 807
(m), 788 (m), 717 (s), 705 (m) cm.sup.-1.
[0167] .sup.1H NMR (CDCl.sub.3, 300 MHz): .delta.=0.88 (t, 3H,
J=6.8 Hz, C17'-H.sub.3), 1.16-1.25 [m, 30H, (C2'-C16')-H.sub.2],
1.85-1.91 (m, 2H, C1'-H.sub.2), 2.23-2.28 (m, 8H, C2-H.sub.2,
C3-H.sub.2, C5-H.sub.2, C6-H.sub.2), 3.69 (s, 6H, OCH.sub.3).
[0168] .sup.13C NMR (CDCl.sub.3, 300 MHz): .delta.=14.7, 23.3,
24.1, 29.1, 29.8, 29.9, 30.0(5), 30.1(2), 30.3, 30.9, 32.5, 36.0,
52.5, 93.3, 173.0.
[0169] MS (EI): m/z=489 (M+NH.sub.4).sup.+.
[0170] MS (EI): m/z (%)=440 [(M-OCH).sup.+, 9], 425 (28), 393
(100), 392 (83), 364 (19), 333 (18), 305 (14), 194 (11), 168 (42),
138 (82), 109 (35), 81 (53).
[0171] HRMS: m/z calcd for C.sub.25H.sub.16NO.sub.5 440.3376
(M-OCH.sub.3).sup.+. Found 440.3379.
[0172] Anal. Calcd for C.sub.26H.sub.49NO.sub.6: C, 66.21; H,
10.47; N, 2.97. Found: C, 66.63; H, 10.91; N, 2.71.
[0173] Dimethyl
3-[(all-Z)-Nonadeca-4,7,10,13-tetraenyl]-3-nitropentane-1,-
5-dicarboxylate
[0174] Following the procedure described above for synthesis of
dimethyl 3-heptadecyl-3-nitropentane-1,5-dicarboxy late (7a),
(all-Z)-1-nitro-5,8,11,14-eicosatetraene (4b) (96 mg, 0.30 mmol)
gave dimethyl
3-[(all-Z)-nonadeca-4,7,10,13-tetraenyl]-3-nitropentane-1,5-dica-
rboxylate (7b) (127 mg, 86%) as a colourless oil.
[0175] IR (film): .nu.=3012 (m), 2935 (m), 2929 (m), 2857 (m), 1742
(s), 1540 (s), 1438 (m), 1379 (w), 1351 (m), 1321 (m), 1260 (m),
1200 (m), 1176 (m), 990 (w), 721 (w) cm.sup.-1.
[0176] .sup.1H NMR (CDCl.sub.3, 300 MHz): .delta.=0.88 (t, 3H,
J=6.8 Hz, C19'-H.sub.3), 1.25-1.35 (m, 8H, C2'-H.sub.2,
C16'-H.sub.2, C17'-Hz, C18'-H.sub.2), 1.86-1.92 (m, 2H,
C1'-H.sub.2), 2.03-2.10 (m, 4H, C3'-H.sub.2, C15'-H.sub.2),
2.25-2.37 (m, 8H, C2-H.sub.2, C3-H.sub.2, C5-H.sub.2,
C.sub.6H.sub.2), 2.78-2.86 (m, 6H, C6'-Hz, C9'-H.sub.2,
C12'-H.sub.2), 3.69 (s, 6H, OCH.sub.3), 5.31-5.43 (m, 8H, C4'-H,
C5'-H, C7'-H, C8'-H, C10'-H, C11'-H, C13'-H, C14'-H).
[0177] .sup.13C NMR (CDCl.sub.3, 300 MHz): .delta.=14.6, 23.1,
24.1, 26.2, 27.4, 27.8, 29.1, 29.9, 30.9, 32.1, 35.4, 52.6, 93.2,
128.1, 128.3, 128.5, 128.9, 129.1, 129.2, 129.9, 131.1, 172.9.
[0178] MS (EI): m/z (%)=491 (M.sup.+, 16), 460 (72), 444 (50), 429
(28), 413 (70), 393 (42), 381 (28), 357 (36), 333 (14), 301 (50),
207 (26), 181 (32), 164 (34), 150 (40), 133 (40), 121 (50), 106
(71), 93 (86), 80 (78), 79 (100), 67 (98), 55 (60).
[0179] HRMS: m/z calcd for C.sub.28H.sub.15NO.sub.6 491.3247
(M.sup.+). Found 491.3247.
[0180] Anal. Calcd for C.sub.28H.sub.45NO.sub.6: C, 68.40; H, 9.22;
N, 2.85. Found C, 68.77; H, 9.57; N, 2.85.
[0181] 3-Heptadecyl-3-nitropentane-1,5-dicarboxylic Acid (8a);
Typical Procedure
[0182] Dimethyl 3-heptadecyl-3-nitropentane-1,5-dicarboxylate (7a)
(138 mg, 0.29 mmol) was dissolved in DME (2 mL) and sat. aq LiOH
solution (2 mL) was added. The mixture was let stand for 22 h, then
it was acidified with dilute HCl (10%, 10 mL) and extracted with
EtOAc (2.times.10 mL). The extracts were concentrated under a
stream of dry N.sub.2 and the residue was subjected to flash column
chromatography on silica (EtOAc/petroleum spirit, 15/85) to afford
3-heptadecyl-3-nitropentane-1,5- -dicarboxylic acid (8a) (93 mg,
90%) as a white solid; mp 102.degree. C.
[0183] IR (Nujol): .nu.=3600-2700 (br), 2919 (s), 2852 (s), 1740
(s), 1700 (w), 1652 (w), 1534 (s), 1467 (m), 1454 (m), 1428 (m),
1353 (w), 1323 (m), 1282 (m), 1267 (w), 1234 (m), 1224 (s), 894
(w), 834 (w), 814 (w), 721 (w) cm.sup.-1.
[0184] .sup.1H NMR (CDCl.sub.3, 300 MHz): .delta.=0.88 (t, 3H,
J=6.8 Hz, C17'-H.sub.3), 1.17-1.30 [m, 30H, (C2'-C16')-H.sub.2],
1.85-1.91 (m, 2H, C1'-H.sub.2), 226-2.40 (m, 8H, C1-H.sub.2,
C2-H.sub.2, C4-H.sub.2, C5-H.sub.2).
[0185] .sup.13C NMR (CDCl.sub.3, 300 MHz): .delta.=14.7, 23.3,
23.9, 29.1, 29.4, 29.8, 29.9(0), 29.9(3), 30.0, 30.1, 30.2, 30.3,
32.5, 37.7, 93.8, 179.2.
[0186] US (CI): m/z=461 (M+NH.sub.4).sup.+.
[0187] MS (EI): m/z (%)=426 [(M-OH).sup.+, 1], 397 (3), 379 (68),
377 (70), 359 (56), 350 (28), 332 (42), 323 (56), 305 (30), 168
(77), 157 (100), 138 (56), 129 (56), 111 (58), 97 (58), 81 (58), 71
(64), 57 (68).
[0188] HRMS: m/z calcd for C.sub.24H.sub.44N 426.3219 (M-OH).sup.+.
Found 426.3229.
[0189] Anal. Calcd for C.sub.24H.sub.45NO.sub.6: C, 64.98; H,
10.22; N, 3.16. Found: C, 64.55; H, 10.69; N, 2.81.
[0190]
3-[(all-Z)-Nonadeca-4,7,10,13-tetraenyl]-3-nitropentane-1,5-dicarbo-
xylic Acid (8b)
[0191] Following the procedure described above for synthesis of
3-heptadecyl-3-nitropentane-1,5-dicarboxylic acid (8a), dimethyl
3-[(all-Z)-nonadeca-4,7,10,13-tetraenyl]-3-nitropentane-1,5-dicarboxylate
(7b) (110 mg, 0.22 mmol) gave
3[(all-Z)-nonadeca-4,7,10,13-tetraenyl]-3-n-
itropentane-1,5-dicarboxylic acid (8b) (90 mg, 88%) as a white
solid; mp 50-51.degree. C.
[0192] IR (film): .nu.=3400-2300 (br), 3013 (s), 2955 (s), 2927
(s), 2855 (s), 2734 (m), 2630 (m), 1742 (s), 1714 (s), 1538 (s),
1439 (s), 1353 (s), 1321 (s), 1291 (s), 1231 (s), 1068 (m), 989
(m), 918 (s), 833 (s), 807 (m), 803 (m), 732 (m), 678 (m), 622 (w)
cm.sup.-1.
[0193] .sup.1H NMR (CDCl.sub.3, 300 MHz): .delta.=0.89 (t, 3H,
J=6.9 Hz; C19'-H3), 1.21-1.38 (m, 8H, C2'-H.sub.2, C16'-H.sub.2,
C17'-H.sub.2, C18'-H.sub.2), 1.85-1.91 (m, 2H, C1'-H.sub.2),
2.03-2.09 (m, 4H, C3'-H.sub.2, C15'-H.sub.2), 2.26-2.38 (m, 8H,
C1-H.sub.2, C2-H.sub.2, C4-H.sub.2, C5-H.sub.2), 2.77-2.86 (m, 6H,
C6'-H.sub.2, C9'-H.sub.2, C12'-H.sub.2), 5.25-5.47 (m, 8H, C4'-H,
C5'-H, C7'-H, C8'-H, C10'-H, C11'-H, C13'-H, C14'-H).
[0194] .sup.13C NMR (CDCl.sub.3, 300 MHz): .delta.=14.7, 23.1,
23.9, 26.2, 27.2, 27.8, 29.2; 29.7, 29.9, 32.1, 36.4, 93.4, 128.1,
128.3, 128.4, 128.9 (2C), 129.2, 130.0, 131.1, 178.8.
[0195] MS (EI): m/z (%)=463 (M.sup.+, 16), 446 (4), 416 (24), 397
(6), 365 (4), 343 (8), 305 (6), 278 (10), 245 (12), 231 (12), 217
(14), 203 (22), 192 (20), 177 (56), 164 (42), 157 (38), 145 (30),
138 (50), 119 (54), 106 (72), 93 (82), 91 (76), 80 (72), 79 (100),
69 (46), 67 (98), 55 (64).
[0196] HRMS: m/z calcd for C.sub.26H.sub.41NO.sub.6 463.2934
(M.sup.+). Found 463.2942.
[0197] Anal. Calcd for C.sub.26H.sub.41NO.sub.6: C, 67.36; H, 8.91;
N, 3.02. Found: C, 67.51; H, 9.23; N, 2.92.
[0198] Octadecanal (9a); Typical Procedure
[0199] PCC (6 g, 27.83 mmol) was suspended in CH.sub.2Cl.sub.2 (30
mL), and octadecan-1-ol (1a) (5.02 g, 18.57 mmol) in
CH.sub.2Cl.sub.2 (15 mL) was then rapidly added at r.t. The
solution became briefly homogeneous before the deposition of the
black insoluble reduced reagent. After 2 h, the black mixture was
diluted with five volumes of anhyd Et.sub.2O, the solvent was
decanted, and the black solid was washed twice with Et.sub.2O. The
crude product was isolated by filtration of the organic solutions
through Florisil and concentration of the filtrate under reduced
pressure. Purification by flash column chromatography on silica
(Et.sub.2O/hexane, 4/96) gave octadecanal (9a) (4.02 g, 81%) as a
white solid; mp 43-44.degree. C.
[0200] IR (Nujol): .nu.=2960 (s), 2910 (s), 2850 (s), 2705 (w),
1730 (s), 1460 (s), 1375 (s), 720 (w) cm.sup.-1.
[0201] .sup.1H NMR (CDCl.sub.3, 300 MHz): .delta.=0.88 (t, 3H,
J=6.4 Hz, C18-H.sub.2), 1.28 [m, 28H, (C4-C17)-H.sub.2], 1.58-1.65
(m, 2H, C3-H.sub.2), 2.42 (t, 2H, J=7.3 Hz, C2-H.sub.2), 9.76 (s,
1H, CHO).
[0202] .sup.13C NMR (CDCl.sub.3, 300 MHz): .delta.=14.7, 22.7,
23.3, 29.7, 29.9, 30.0, 30.1, 30.3, 32.5, 44.5, 203.6.
[0203] MS (EI): m/z (%)=268 (M.sup.+, 4), 250 (34), 224 (17), 222
(18), 208 (6), 194 (10), 182 (8), 166 (8), 152 (10), 137 (20), 124
(30), 110 (42), 96 (74), 82 (100), 71 (82), 69 (69), 57 (53), 55
(57).
[0204] HRMS: m/z calcd for C.sub.18H.sub.36O 268.2766 (M.sup.+).
Found: 268.2765.
[0205] Anal. Calcd for C.sub.81H.sub.36O: C, 80.53; H, 13.51.
Found: 80.46, H, 13.49.
[0206] (all-z)-Eicosa-5,8,11,14-tetraenal (9b)
[0207] According to the procedure described above for preparation
of octadecanal (9a), arachidonyl alcohol (1b) (402 mg, 1.38 mmol)
gave (all-Z)-eicosa-5,8,11,14-tetraenal (9b) (303 mg, 76%) as a
colourless oil.
[0208] IR (film): .nu.=3005 (s), 2960 (s), 2910 (s), 2850 (s), 1730
(s), 1460 (w), 1390 (w), 1160 (w), 920 (w) cm.sup.-1.
[0209] .sup.1H NMR (CDCl.sub.3, 300 MHz): .delta.=0.89 (t, 3H,
J=6.8 Hz, C20H), 1.28-1.34 (m, 6H, C17-H.sub.2, C18-H.sub.2,
C19-H.sub.2), 1.69-1.74 (m, 2H, C.sub.3H.sub.2), 2.04-2.14 (m, 4H,
C4-H.sub.2, C16-H.sub.2), 2.42-2.45 (m, 2H, C2-H.sub.2), 2.79-2.85
(m, 6H, C7-H.sub.2, C10-H.sub.2, C13-H.sub.2), 5.345.40 (m, 8H,
C5-H, C6-H, C8-H, C9-H, C11-H, C12-H, C14-H, C15-H), 9.78 (s, 1H,
CHO).
[0210] .sup.13C NMR (CDCl.sub.3, 300 MHz): .delta.=14.5, 22.3,
23.0, 26.1, 26.9, 27.6, 29.7, 31.9, 43.7, 127.9, 128.2, 128.4,
128.7, 129.0, 129.2, 129.5, 130.9, 202.9.
[0211] MS (EI): m/z (%) 288 (M.sup.+, <1), 244 (1), 234 (1), 217
(2), 203 (3), 177 (9), 164 (13), 150 (30), 131 (12), 119 (19), 106
(59), 93 (56), 91 (64), 80 (77), 79 (100), 67 (93), 55 (43).
[0212] HRMS: m/z calcd for C.sub.20H.sub.32O 288.2453 (M.sup.+).
Found: 288.2449.
[0213] Anal. Calcd for C.sub.20H.sub.32O: C, 83.27; H, 11.18.
Found: C, 83.28; H, 11.12.
[0214] 1-Nitrononadecan-2-ol (10a); Typical Procedure
[0215] To a solution of octadecanal (9a) (2.22 g, 8.28 mmol) and
nitromethane (1.52 g, 24.90 mmol) in anhyd Et.sub.2O (10 mL),
Amberlyst A-21 (1.2 g) was added at r.t. The mixture was stirred
and heated at reflux for 48 h. After removal of the Amberlyst A-21
by filtration, the filtrate was concentrated under reduced
pressure. Flash column chromatography of the residue
(EtOAc/petroleum spirit, 5/95) gave 1-nitrononadecan-2-ol (10a)
(2.41 g, 89%) as a white solid; mp 55-56.degree. C.
[0216] IR (Nujol): .nu.=3500-3300 (br), 2960 (s), 2910 (s), 2850
(s), 1550 (m), 1460 (s), 1375 (s), 720 (w) cm.sup.-1.
[0217] .sup.1H NMR (CDCl.sub.3, 300 MHz): .delta.=0.86-0.90 (m, 3H,
C19-H3), 1.26 [m, 30H, (C.sub.4-C18)-H.sub.2], 1.43-1.55 (m, 2H,
C3-H.sub.2), 2.22-2.43 (bs, 1H, OH), 4.28-4.46 (m, 3H, C1-H.sub.2,
C2-H).
[0218] .sup.13C NMR (CDCl.sub.3, 300 MHz): .delta.=14.7, 23.3,
25.7, 29.8(8), 29.9(2), 30.0, 30.1, 30.2, 30.3, 32.5, 34.3, 69.2,
81.2.
[0219] MS (CI): m/z=347 (M+NH.sub.4).sup.+.
[0220] MS (EI): m/z (%)=311 [(M-H.sub.2O).sup.+, 3], 294 (32), 282
(9), 276 (27), 267 (31), 250 (34), 240 (6), 272 (15), 208 (8), 194
(9), 179 (7), 165 (10), 151 (16), 137 (37), 123 (62), 109 (85), 97
(95), 95 (100), 83 (100), 69 (88), 57 (92), 55 (92).
[0221] HR: m/z calcd for C.sub.19H.sub.37NO.sub.2 311.2824
(M-H.sub.2O).sup.+. Found: 311.2831.
[0222] Anal. Calcd for C.sub.19H.sub.39NO.sub.3: C, 69.25; H,
11.93, N, 4.25. Found: C, 69.54, H, 12.18, N, 4.13.
[0223] (all-Z)-Nitroheneicosa-6,9,12,15-tetraen-2-ol (10b)
[0224] According to the procedure described above for synthesis of
1-nitrononadecan-2-ol (10a), (all-Z)-eicosa-5,8,11,14-tetraenal
(9b) (220 mg, 0.76 mmol) gave
(all-Z)-1-nitroheneicosa-6,9,12,15-tetraen-2-ol (10b) (240 mg, 90%)
as a colourless oil.
[0225] IR (film): .nu.=3600-3300 (br), 3005 (s), 2960 (s), 2910
(s), 2850 (s), 1650 (w), 1550 (s), 1460 (m), 1440 (m), 1380 (s),
1260 (w), 910 (w), 720 (s) cm.sup.-1.
[0226] .sup.1H NMR (CDCl.sub.3, 300 MHz): .delta.=0.87-0.91 (m, 3H,
C21-H.sub.3), 1.27-1.39 (m, 6H, C18-H.sub.2, C19-H.sub.2,
C20-H.sub.2), 1.50-1.56 (m, 4H, C3-H.sub.2, C.sub.4H.sub.2),
2.02-2.16 (m, 4H, C5-H.sub.2, C17-H.sub.2), 2.40-2.60 (bs, 1H, OH),
2.80-2.86 (m, 6H, C8-H.sub.2, C11-H.sub.2, C14-H.sub.2), 4.294.45
(m, 3H, C1-H.sub.2, C2-H), 5.30-5.45 (m, 8H, C6-H, C7-H, C9-H,
C10-H, C12-H, C13-H, C15-H, C16-H).
[0227] .sup.13C NMR (CDCl.sub.3, 300 MHz): .delta.=14.5, 23.0,
25.5, 26.0, 27.1, 27.6, 29.7, 31.9, 33.5, 68.9, 81.0, 127.9, 128.2,
128.5, 128.6, 129.0, 129.1, 129.5, 130.9.
[0228] MS (EI): m/z (%)=349 (M.sup.+, <1), 314 (1), 251 (2), 234
(1), 217 (2), 203 (3), 177 (6), 164 (10), 150 (24), 131 (13), 119
(21), 106 (43), 93 (57), 91 (71), 79 (100), 67 (92), 55 (48).
[0229] HRMS: m/z calcd for C.sub.21H.sub.35NO.sub.3 349.2617
(M.sup.+). Found: 349.2614.
[0230] Anal. Calcd for C.sub.21H.sub.35NO.sub.3: C, 72.17; H,
10.09, N, 4.01. Found: C, 72.25, H, 9.91; N, 3.64.
[0231] B. Determination of Biological Activity of Nitro Compounds
[4a (Lx1); 4b (Lx4); 6a (Lx6); 6b (Lx7); 8a (Lx8) and 8b (Lx9)]
[0232] (1) Investigation of 15-LO, 5-LO and 12-LO Catalysed
Oxidation of the Nitro Compounds (4a, 4b, 6a, 6b, 8a and 8b;Table
1)
[0233] It has been suggested the various hydroxy and hydroperoxy
fatty acid derivatives (such as 15-HETE and 15 HPETE) have
inhibitory effects on lipoxygenase enzymes..sup.[35] Based on this
consideration, 5-LO, 12-LO and 15-LO catalysed oxidation of the
nitro compounds (4a, 4b, 6a, 6b, 8a and 8b) was investigated. Each
of the nitro compounds was treated with 15-LO in pH 9.0 buffer (or
5-LO in pH 6.3 buffer and 12-LO in pH 7.4 buffer), and the
formation of 15-hydroperoxy derivatives (or 5-hydroperoxy or
12-hydroperoxy derivatives) over time was monitored by UV
spectroscopy at 234 nm. The result shows that, among the nitro
compounds, compound 6b was the only one that underwent lipoxygenase
catalysed oxidation. It served as a substrate for both 15-LO and
12-LO, but not for 5-LO.
[0234] (2) The Effect of Nitro Compounds 4a (Lx1), 4b (Lx4), 6a
(Lx6), 6b (Lx7), 6a (Lx8) and 8b (Lx9) on 15-LO, 5-LO and 12-LO
Catalysed Oxidation of Arachidonic Acid
[0235] The result from the preliminary experiment is summarised in
Table 2. It shows that compound 8a has an inhibitory effect on
15-LO but not on 5-LO, while compound 6a displays complementary
activity inhibiting 5-LO but not 15-LO. Neither 8a nor 6a inhibits
12-LO. Compound 8b appears to have a significant inhibitory effect
on 12-LO catalysed oxidation of arachidonic acid, giving a
relatively long lagtime at the early stage of arachidonic acid
oxidation.
[0236] (3) The Inhibitory Effect of 15-hydroperoxy and 15-hydroxy
Derivatives from Compound 6b on 15-LO Catalysed Oxidation of
Arachidonic Acid
[0237] An enzyme assay shows that these two compounds did have
inhibitory effect on 15-LO catalysed oxidation of arachidonic acid,
giving IC.sub.50 values of 50 .mu.M for 15-hydroperoxy derivative
of 6b and 120 .mu.M for 15-hydroxy derivative of 6b.
[0238] (4) Determination of K.sub.m and V.sub.max for 15-LO
Catalysed Oxidation of Compound 6b, and Inhibitor Constant of
Compound 8a on 15-LO Catalysed Oxidation of Arachidonic Acid
[0239] The Michaelis constant K.sub.m and the value of V.sub.max
for 15-LO catalysed oxidation of compound 6b were measured and
calculated based on the Lineweaver Burke equation, with K.sub.m as
8.4 .mu.M and V.sub.max as 24.48 .mu.M/min.
[0240] The inhibitor constant (K.sub.i or K.sub.1) of compound 8a
was also determined. The graph of 1/v vs 1/[s] with varying
concentrations of compound 8a indicates that the inhibition is of
the mixed inhibition pattern as shown in the following scheme. Thus
the K.sub.i and K.sub.1 values in the scheme were calculated giving
the result of 27.42 .mu.M for K.sub.i and 55.15 .mu.M for
K.sub.1.
2TABLE 2 Effect of nitro compounds on oxidation of arachidonic acid
(AA) catalysed by 15-LO, 5-LO or 12-LO Effect on 12-LO Effect on
15-LO Effect on 5-LO catalysed catalysed catalysed oxidation
Compounds oxidation of AA oxidation of AA of AA Lx1 Nil Nil Nil Lx4
Nil Nil Nil Lx6 Activatory Inhibitory Activatory IC.sub.50 = 60
.mu.M Lx8 Inhibitory Nil Activatory K.sub.i = 27.42 .mu.M K.sub.I =
55.15 .mu.M Lx7 Substrate Activatory Substrate K.sub.m = 8.4 .mu.M
V.sub.max = 24.48 .mu.M/min Lx9 Nil Activatory Inhibitory Lx2 Nil
Nd Nd Lx3 Nil Nd Nd Lx5 Nil Nd Nd Nd = Not done
[0241] C. Anti-Tumour Effects of Nitro Compounds
[0242] (1) Prostate Cancer
[0243] Prostate cancer is the most often diagnosed non-skin cancer
and second largest cause of cancer related death in men in the
United States.sup.(36). Frostate cancer usually commences as an
androgen-dependent cancer which responds well to treatments such as
hormone ablation therapy. However, the cancer can progress to an
androgen-independent form which is usually fatal.sup.(37). The
prognosis for prostate cancer is so poor that the Urological
Society of Australasia has stated that there is no point
undertaking population screening until there are viable treatments
available.sup.(38). Therefore, there is an urgent need for new
therapies to treat androgen-independent prostate cancer; ideally
therapies which induce apoptosis in androgen-independent prostate
cancer cells.
[0244] As humans are one of the few species to get prostate cancer,
whole animal work has been limited. This situation has recently
changed with the release of a mouse genetically-engineered to
develop prostate cancer.sup.(39). However, most research work on
prostate cancer so far has been done using human-derived tissue
culture cell lines. The three most commonly used cell lines are
LNCaP, DU145 and PC3 which were derived from humans with metastases
to lymph node, brain and bone respectively. LNCaP cells are
androgen sensitive in that the addition of androgens can cause a
biological response, eg growth modulation.sup.(40), but they are
not androgen-dependent as they do not die following withdrawal of
androgen and can continue to proliferate in the absence of
androgen. DU145 and PC3 cells are androgen-independent as they do
not respond to the addition or withdrawal of androgen. DU145 and
PC3 cells are generally more resistant to inducers of apoptosis
than LNCaP. For example, the phorbol ester, phorbol 12-myristate
13-acetate, induces apoptosis in LNCaP but not DU145
cells.sup.(41). The increased resistance of androgen-independent
cell lines mirrors the clinical situation where
androgen-independent cancer is resistant to treatment and is
usually fatal.
[0245] Although the aetiology of prostate cancer is still poorly
understood, there is evidence that dietary fat intake can influence
prostate cancer risk. While intake of high levels of the n-6 fatty
acid, arachidonic acid (20:4n-6), promotes the growth of prostate
and breast cancers, increased intake of n-3 fatty acids such as
eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3),
found in abundance in fish oils, reduces the risk of these
cancers.sup.(42). An analysis of the ratio of n-3:n-6 levels in the
serum of patients with prostate cancer and benign prostate
hyperplasia, and age-matched controls, has revealed that the
patients have a lower n-3:n-6 fatty acids compared to
controls.sup.(43). Another study has found that reduced prostate
cancer risk is associated with increased levels of 20:5n-3 and
22:6n-3 esterified in phosphatidylcholine in cellular
membranes.sup.(44). These studies therefore suggest that the
polyunsaturated fatty acids (PUFA) or their metabolites are
important regulators of prostate cancer development and growth.
[0246] Fatty acids are essential components of cellular membranes
and are an important source of fuel. Furthermore, fatty acids,
especially PUFA such as 20:4n-6, 20:5n-3 and 22:6n-3, are
biologically active when added exogenously to a variety of
cell-types. The actions of these PUFA range from stimulation of
neutrophil responses to inhibition of cell-cell communication via
gap junctions.sup.(45,46). n-6 and n-3 PUFA have also been
demonstrated to stimulate the activities of protein kinase C and
MAP kinases such as the extracellular signal-regulated protein
kinase, c-jun N-terminal kinase and p38, and to cause an increase
in intracellular Ca.sup.2+ concentration.sup.(47-50). Inhibitor
studies have indicated that some of the actions of the PUFA are
mediated by these intracellular signalling molecules.sup.(50). The
actions of the PUFA can also be mediated by their metabolites
derived from the 5-, 12- and 15-lipoxygenases (LOX), and
cyclooxygenases (COX) 1 and 2. For example, 5-LOX-derived
eicosanoids, 5-oxo eicosatetraenoic acid and leukotriene B4 are
potent activators of neutrophils and eosinophils.sup.(51). PUFA can
also influence cellular responses by being incorporated into
membrane phospholipids which serve as substrates for phospolipases
A.sub.2, C and D, thereby giving rise to important lipid-based
second messenger molecules such as non-esterified fatty acids,
diacylglycerol, phosphatidic acid and lysophosphatidic acid.
[0247] The reasons for the above described antithetic actions of
n-3 and n-6 PUFA on prostate cancer risk have not been elucidated.
Since 20:5n-3 PUFA compete against 20:4n-6 as substrates for 5-LOX,
it is possible that a reduction in eicosanoids derived from 20:4n-6
in the presence of 20:5n-3 may have an impact on the survival of
prostate cancer cells. Similarly, such competition between n-3 and
n-6 PUFA for metabolism by COX may also be relevant. If this is
correct, inhibition of the 5-LOX or COX will result in the death of
prostate cancer cells. Interestingly, it has recently been
demonstrated that apoptosis can indeed be induced in prostate
cancer cell lines using inhibitors of 5-LOX such as NDGA and
MK886.sup.(52,53) or COX such as ibuprofen.sup.(54). For 5-LOX to
be active, it must be bound to 5-LOX activating protein
(FLAP).sup.(55). MK886 works by binding to FLAP while NDGA is a
broad inhibitor of lipoxygenase activity. The effect of 5-LOX
inhibitors can be reversed by the addition of
5-hydroxyeicosatetraenoic acid, but not by 12- or
15-hydroxyeicosatetraenoic acid.sup.(33). This provides strong
evidence that (a) product(s) of 5-LOX is (are) needed for the
survival of prostate cancer cells. The apoptosis-inducing action of
5-LOX inhibitors is not restricted to prostate cancer cell lines,
as inhibition of 5-LOX has also been shown to induce apoptosis in a
number of lung cancer cell lines and in in vivo models of lung
cancer.sup.(56), These results suggest that 5-LOX may be a target
for anti-cancer drugs. Besides the Lx compounds described above, we
have also previously synthesized oxa and .beta.-thia fatty acids
(MN) (International Patent Specification No. PCT/AU95/00677) and
fatty acid-amino acid conjugates (PT) (International Patent
Specification No. PCT/AU95/00717) (Table 3). Our investigations
with some of these compounds have revealed that a number of them
are strong inhibitors of purified 5-LOX and 5-LOX catalysed
production of leukotriene 134 in neutrophils but not of COX in
these cells. Others alter the LOX differently. Thus an examination
of the ability of these PUFA to kill cancer cells was
undertaken.
3TABLE 3 Synthetic fatty acids and related compounds. Amino acid-
.beta.-oxa PUFA .beta.-and .gamma.-thia PUFA conjugated PUFA MP1
(.beta.-oxa-23:0) MP1 (.beta.-thia 23:0) PT7 (18:3n-6-Gly) MP4
(.beta.-oxa-21:3n-6) MP9 (.beta.-thia-21:3n-6) PT8 (18:3n-6-Asp)
MP5 (.beta.-oxa-21:3n-3) MP10 (.beta.-thia-21:3n-3) PT9
(18:3n-3-Gly) MP7 (.beta.-oxa-21:4n-3) MP8 (.beta.-thia-23:4n-6)
PT10 (18:3n-3-Asp) MP3 (.beta.-oxa-23:4n-6) MP12
(.gamma.-thia-22:3n-6) PT1 (20:4n-6-Gly) MP6 (.beta.-oxa-25:6n-3)
MP13 (.gamma.-thia-22:3n-3) PT2 (20:4n-6-Asp) MP11
(.gamma.-thia-24:4n-6) PT3 (20:5n-3-Gly) MP14
(.gamma.-thia-25:6n-3) PT4 (20:5n-3-Asp) MP15
(.alpha.-CH.sub.2CO.sub.2H-.beta.-thia PT5 (22:6n-3-Gly) 23:4n-6)
PT6 (22:6n-3-Asp) MP16 (15-OOC(CH.sub.3).sub.2 OCH.sub.320:4n-6)
MP17 (15'-OOC(CH.sub.3).sub.2 OCH.sub.3 .beta.-oxa 23:4n-6)
Protected hydroperoxy PUFA Nitroso-compounds MP16
(15-OOC(CH.sub.3).sub.2OCH.sub.3 LX1 (19:0-NO.sub.2) 20:4n-6) LX2
(19:3n-3-NO.sub.2) MP17 (15'-OOC(CH.sub.3).sub.2OCH.sub.1 LX3
(19:3n-6-NO.sub.2) .beta.-oxa-23:4n-6) LX4 (21:4n-6-NO.sub.2)
Hydroxy.beta.-oxa-PUFA LX5 (23:6n-3-NO.sub.2) TR1 (16-OH
.beta.-oxa-21:3n-6) LX6 (21:0-.gamma.-NO.sub.2) TR2 (16-OH
.beta.-oxa-21:3n-3) LX7 (23:4n-6-.gamma.-NO.sub.2) LX8
(.gamma.,.gamma.-COOH-19:0-NO.sub- .2) LX9
(.gamma.,.gamma.-COOH-21:4n-6-NO.sub.2)
[0248] Lx compounds were tested for activity against two androgen
insensitive prostate cancer tumour cell lines, DU145 (liver
metastases) and PC3 (brain metastases). The compounds showed anti
tumour effects (FIGS. 1,2,3). FIG. 1 shows the survival of DU145 of
Lx compound. Survival was measusred using the MTS cell
proliferation assay.
[0249] Various concentrations for varying amounts of time of Lx
compounds were added to either DU145 or PC3 tumour cells in
culture. Viability/death of tumour cells was measured by a standard
colourimetric assay. The results in FIG. 2 show both the
concentration and time related effects of Lx6 on DU145 tumour
cells. Using a concentration of 15 .mu.M, killing of all the tumour
cell population occurred after 24 hours of culture. It is also
evident from the results (FIG. 2) that the saturated nitro compound
Lx1 (19:0-NO.sub.2) is not active and neither is MP2 (.beta.-thia
23:0), a saturated .beta.-oxa fatty acid. Using the PC3 tumour cell
line, similar results were found (FIG. 3).
[0250] In a further screening test, the human prostate cell line,
DU145 cells (liver metastases), was treated for 24 hrs with 20
.mu.M Fatty Acid and cell survival was measured using the MTS cell
proliferation assay. This assay uses colourimetric measurement of
substrate conversion to formazan which occurs only in the presence
of NADH in a metabolically active cell. The results are presented
in FIG. 4. There was varied anti-tumour activity amongst the
different types of PUFA. Of the MP compounds, MP6
(.beta.-oxa-25:6n-3), MP9 (.beta.-thia-21:3n-6), MP10
(.beta.-thia-21:3n-3), MP12 (.gamma.-thia-22:3n-6) and MP17
(15'-OOC[CH.sub.3].sub.2CH.sub.3 .beta.-oxa-23:4n-6) were the most
active in killing DU145 cells. In addition, MP3
(.beta.-oxa-23:4n-6), MP8 (A-thia-23:4n-6) and MP13
(.beta.-thia-22:3n-3) were also highly active. In the PT series,
PT5 (22:6n-3 Gly) showed some activity. These studies show that the
anti-cancer activity of the PUFA is dependent on their structure.
Using the PC3 cell line (12 metastases), similar results were
found.
[0251] To see whether the method by which the fatty acids killed
tumour cells was by apoptosis, we measured the activation of
caspases. DU145 cells were treated with PUFA and incubated for 24 h
(for the proliferation assay), 4 h (for the caspase assay) and for
18 h (for PARP cleavage).
[0252] Proliferation was quantitated by MTS assay. Caspases 3 and 7
were assayed by a fluorometric assay using a DVED substrate. PARP
cleavage was measured by Western blots using an anti PARP antibody.
The results showed that, under conditions where the PUFA, MP3 and
MP5 caused marked inhibition of cell proliferation (FIG. 5A), there
was activation of caspases in association with the cleavage of PARP
(FIGS. 5B and 5C). MP3 and MP5, but not MP1, induce apoptosis in
DU145 cells.
[0253] (2) Breast Cancer
[0254] Breast cancer is the most commonly diagnosed cancer and the
main cause of cancer-related death in women in Australia.sup.(57).
This is in contrast to Japan, where breast cancer is rare even
though Japanese women living in western countries have the same
incidence rates as western women.sup.(58). This has given rise to
the hypothesis that environmental factors can affect breast cancer
risk. One environmental difference which has generated a lot of
interest is diet. The Japanese diet contains more fish than the
western diet and fish contains high levels of n-3 fatty acids.
[0255] There has been some research into the effect of n-3 fatty
acids on breast cancer. While intake of high levels of the n-6
fatty acid, arachidonic acid (20:4n-6), promotes the growth of
prostate and breast cancers, increased intake of n-3 fatty acids
such as eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid
(22:6n-3), found in abundance in fish oils, reduces the risk of
these cancers.sup.(59). n-3 fatty acids have also been shown to
inhibit metastasis of human breast cancer xenografts in mice
whereas n-6 fatty acids promoted metastasis.sup.(60). These studies
suggest that the polyunsaturated fatty acids (PUFA) or their
metabolites are important regulators of breast cancer development
and growth.
[0256] As has been discussed for prostate cancer, it is possible
that a reduction in eicosanoids derived from 20:4n-6 in the
presence of n-3 PUFA may have an impact on the survival of breast
cancer cells. Similarly, such competition between n-3 and n-6 PUFA
for metabolism by COX may also be relevant. If this is correct,
then interference with metabolism of 20:4n-6 by LOX or COX will
result in the death of breast cancer cells.sup.(61).
[0257] Interestingly, it has been reported that breast cancer cells
are dependent on 5-LOX and 12-LOX but in different ways. Addition
of 5-LOX to MCF-7 breast cancer cells inhibits their growth and the
5-LOX inhibitor MK886 can reverse 5-LOX growth inhibition.sup.(62).
Therefore, the 5-LOX derived metabolites must be
anti-proliferative. In contrast, expression of 12-LOX in MCF-7
cells enhances growth.sup.(63). Furthermore, breast cancer biopsies
and cell lines have increased expression of 12-LOX mRNA compared to
benign breast tissue and cell lines.sup.(64). Therefore, products
of 12-LOX must stimulate breast cancer growth. These results
suggest that activation of 5-LOX and inhibition of 12-LOX may be a
means of treating breast cancer.
[0258] A number of Lx compounds were tested for anti 5-LOX and
12-LOX activities in the human breast cancer cell line, MCF-7. The
results in FIG. 6 show that the Lx compounds exhibit inhibitory
effects against 5-LOX and 12-LOX activities. The ability of 5-LOX
and 12-LOX to produce oxidated products of 20:4n-6 with a different
absorption wavelength in the presence of nitroso-PUFA was examined
and is shown in the progress curves in FIG. 6. Both Lx7 and Lx9 can
activate 5-LOX (FIGS. 6A and 6B) in a purified enzyme system.
Within cells, activation of 5-LOX is facilitated by 5-LOX
activating protein (FLAP). Thus, in intact cells, Lx7 and Lx9 may
be able to activate 5-LOX at lower concentrations than those
required in the purified enzyme system shown in FIG. 6. Lx7 is also
a substrate for 12-LOX (FIG. 6C) and may compete with 20:4n-6 in
vivo resulting in the loss of 12-LOX growth promoting products
whereas Lx9 is a direct, potent and rapid inhibitor of 12-LOX (FIG.
6D). By being able to activate 5-LOX and suppress 12-LOX, these
compounds have the desired characteristics of anti-breast cancer
agents as discussed previously.
[0259] While interference with 5-LOX or 12-LOX activity
individually impacts on growth rates of human breast cancer MCF-7
cells.sup.(55,56), the simultaneous modification of the activities
of both enzymes at once may cause death. When Lx7 and Lx9 were
added to MCF-7 cells in nitro, these two compounds were found to
kill these tumour cells (FIG. 7) at concentrations below those at
which 20:4n-6 became toxic. Lx4, a nitroso-compound with the same
number of carbon atoms and degree of unsaturation as Lx9 but
lacking a COOH group, was ineffective (FIG. 7).
[0260] D. Properties of the .beta. and .gamma. Oxa and Thia Fatty
Acids
[0261] Other analogues of PUFAs targeted in this project were the
oxa and thia fatty acids, owing to their potential as antioxidants,
and therefore corresponding potential as anti-cancer agents.
Compounds of types 16-19, as identified in Table 4, were
constructed as PUFA analogues having the property of resistance to
.beta.-oxidation.sup.(67,13).
4TABLE 4 Structure and nomenclature of the oxa and this fatty acid
analogues and other thia compounds Structure Systematic name WCH
Thesis 26 (Z,Z,Z)-(octadeca-6,9,12-trienyloxy)acetic acid 16 MP4 27
(Z,Z,Z)-(octadeca-9,12,15-trienyloxy)acetic acid 17 MP5 28
(all-Z)-(eicosa-5,8,11,14-tetraenylthio)acetic acid 18 MP8 29
3-[(all-Z)-(eicosa-5,8,11,14-tetra- enylthio)propionic acid 19 MP11
30 3-[(3Z,6Z)-nona-3,6-dienylthiopropionic acid 106 31
3-tetradecylthiopropionic acid 108 32 2-tetradecylthiopropionic
acid 109 33 propyl(all-Z)-eicosa-5,8,11,14-tetraenylpropyl sulfide
110 34 propyltetradecyl sulfide 111 35
3-[(Z,Z,Z)-(octadeca-9,12,15-trienyl- thio)]propionic acid 112 MP13
36 3-(tetradecylsulfinyl)propionic acid 113 37
2-(tetradecylsulfinyl)acetic acid 114
[0262] Experimental
[0263] .sup.1H NMR and .sup.13C NMR spectra were recorded on a
Gemini 300 MHz or a Unity Inova 500 MHz spectrometers with
tetramethylsilane (TMS) as the internal standard (.delta.0.00 ppm).
Samples were run in deuterochloroform (99.8% D) unless indicated
otherwise. The following abbreviations are adopted: s (singlet); d
(doublet); t (triplet); m (multiplet); dd (doublet of doublets); bs
(broad singlet). J values are given in Hz.
[0264] Infrared (IR) spectra were recorded on Perkin-Elmer 683 and
7700 infrared spectrophotometers. The following abbreviations are
used: br (broad), w (weak), m (medium), s (strong).
[0265] Ultraviolet spectra were recorded on a Shimadzu UV 2101 PC
spectrophotometer with a temperature controller and kinetic
software.
[0266] Low and high resolution electron ionisation (EI) mass
spectra and chemical ionisation (CI) mass spectra were run on a
Fisons VG Autospec. A Fisons VG Instrument Quattro II mass
spectrometer was used for negative ion electrospray mass spectra.
Gas chromatography-mass spectrometry (GC-MS) was carried out with a
HP 5970 mass selective detector connected to a HP 5890 gas
chromatography with a 12.5 m BP-1 column.
[0267] Melting points were determined using a Reichert microscope
with a Kofler heating stage and are uncorrected. Buffers were
adjusted to the required Ph using a model 520A pH meter.
Microanalyses were conducted by the Microanalytical Laboratory,
Research School of Chemistry, Australian National University.
[0268] HPLC was performed using a Waters HPLC system with
ultraviolet (UV) or refractive index (RI) detection. The column
used contained Alltech Spherisorb octadecylsilane (ODS) (1.6
mm.times.250 mm, 3 .mu.m). The mobile phase was comprised of
acetonitrile (or methanol) and phosphoric acid (30 mM) solution in
the ratios indicated in the text, with a flow rate of 1 nm/min.
[0269] Column chromatography was carried out using Merck Silicagel
60 as the absorbent. Analytical TLC was performed on Merck
Silicagel 60 F.sub.254 silica on aluminium baked plates.
[0270] 15-LO was obtained from Sigma Chemical Company, and 12-LO
from Cayman Chemical Company. Arachidonic acid 1, linolenyl alcohol
57a, gamma linolenyl alcohol 57b, arachidonyl alcohol 57c and
docosahexaenyl alcohol 57d were purchased from Nu-Chek Prep. Inc.
Elysian, Minn., USA. Other chemicals were commercially available
from Aldrich Chemical Company.
[0271] Determination of Stability of Thia Fatty Acids and
Sulfides
[0272] Compounds 110 (4.3 mg) and 106 (6 mg) were each dissolved in
5 ml of dichloromethane and added into 250 ml one-neck flasks.
Compound 18 (20 mg) and compounds 19, 108, 109 and 111-112 (14-20
mg) were each dissolved in 10 ml of dichloromethane and added into
500 ml flasks. The solvent dichloromethane was then evaporated with
continuous rotation of the flasks, allowing the compounds to form
thin films. The flasks were flushed with oxygen, sealed and kept in
darkness.
[0273] The compounds in the flasks were redissolved in chloroform-d
and analysed by .sup.1H NMR every two weeks for up to six
weeks.
[0274] Determination of Antioxidant Behaviour of
3[(3Z,6Z)-nona-3,6-dienyl- thio]propionic Acid on Arachidonic Acid
Autoxidation
[0275] This is a typical autoxidation assay designed to investigate
the antioxidant properties of thia fatty acids and sulfides in the
autoxidation of arachidonic acid 1.
[0276] A stock solution in dichloromethane (2 ml) containing
arachidonic acid 1 (18 mg) and
3-[(3Z,6Z)-nona-3,6-dienylthio]propionic acid 106 (18 mg) was
prepared with lauric acid (18 mg) as an internal standard. Samples
of the stock solution (100 .mu.l) were added to glass Petri-dishes
followed by ethanol (400 .mu.l). After evaporation of the solvent,
a well-distributed thin film was formed on each Petri-dish. The
Petri-dishes were placed in a desiccator, which was then evacuated,
filled with oxygen and stored in the darkness. Dishes were removed
from the desiccator after 1, 2, 3, 5 and 7 days. The mixture on
each dish was redissolved in diethyl ether and transferred to a 2
ml vial. After evaporation of the solvent, the residue was
dissolved in the HPLC mobile phase (100 .mu.l) and 10% of the
solution was analysed by HPLC using a reverse phase column
(octadecylsilane) (4.6 mm.times.250 nun, 3 .mu.m) and a refractive
index detector. Table 5 shows the mobile phases used for different
thia fatty acids and sulfides, and their retention times by
HPLC.
5TABLE 5 HPLC mobile phase and retention time of thia fatty acids
and sulfides Retention Retention Mobile phase time (min) time (min)
Retention (Buffer = 30 mM (Arachidonic (Lauric time (min) Compound
H.sub.3PO.sub.4) acid 1) acid) (Compound) 18 Acetonitrile- 6.53
4.23 8.75 Buffer (80:20) 19 Acetonitrile- 6.80 4.44 10.91 Buffer
(80:20) 106 Acetonitrile- 14.71 7.13 3.15 Buffer (70:30) 108
Methanol-Buffer 6.71 4.00 10.74 (90:10) 109 Methanol-Buffer 6.82
4.05 9.38 (90:10) 110 Acetonitrile- 3.48 3.09 14.05 Buffer (95:5)
111 Acetonitrile- 3.38 3.05 21.57 Buffer (95:5) 112 Acetonitrile-
5.24 3.80 6.97 Buffer (90:10)
[0277] Synthesis of analogues of
3-[(all-Z)-(eicosa-5,8,11,14-tetraenyl-th- io)]propionic Acid
[0278] Pent-2-ynyl p-toluenesulfonate, 102. 2-Pentyn-1-ol 101 (1.03
g, 12 mmol) was dissolved in chloroform (10 ml) and the mixture was
cooled in an ice bath Pyridine (1.90 g, 24 mmol, 2 eq) was then
added, followed by p-toluenesulfonyl chloride (3.43 g, 18 mmol, 1.5
eq) in small portions with constant stirring. The reaction was
complete in 4 h (monitored by TLC). Ether (30 ml) and water (7 ml)
were added and the organic layer was washed successively with 1 N
HCl (7 ml), 5% NaHCO.sub.3, water (7 ml) and brine (20 ml), and
then dried with Na.sub.2SO.sub.4. The solvent was removed under
reduced pressure and the crude tosylate was flash column
chromatographed on silica gel using ether-hexane (20:80) as the
eluent to yield the title compound 102 (1.85 g, 65%) as a
colourless oil. Found: C, 60.24; H, 5.93; S, 13.22. Calc. for
C.sub.12H.sub.14SO.sub.3: C, 60.48; H, 5.92; S, 13.45%.
.nu..sub.max (film)/cm.sup.-1 2980 (m), 2940 (w), 2878 (w), 2240
(m), 1598 (s), 1495 (w), 1450 (m), 1360 (s), 1180 (s), 1175 (s),
1095 (s), 1020 (m), 1000 (m), 960 (s), 940 (s), 840 (s), 815 (s),
735 (s), 662 (s); BH (300 MHz, CDCl.sub.3) 0.98-1.03 (3H, m,
C5-H.sub.3); 2.04-2.10 (2H, m, C4-H.sub.2), 2.44 (3H, s,
ArCH.sub.3), 4.69 (2H, m, C1-H.sub.2), 7.35 and 7.82 (4H, dd, J 8.3
and 8.7, ArH); .delta..sub.C (300 MHz, CDCl.sub.3) 12.91, 13.72,
22.22, 59.35, 71.72, 92.33, 128.69, 130.30, 133.90, 145.47; m/e
(EI): 238 (M.sup.+, <0.1% o), 209 (1), 155 (24), 139 (100), 129
(6), 117 (18), 107 (10), 92 (42), 91 (87), 83 (29), 66 (30), 65
(48).
[0279] Nona-3,6-diyn-1-ol, 103. Pent-2-ynyl p-toluenesolfonate 102
(1.37 g, 5.78 mmol, 1.1 eq) was added at -30.degree. C. under
nitrogen to a well-stirred suspension in DMF (15 ml) of
but-3-yn-1-ol (368 mg, 5.25 mmol, 1 eq), sodium carbonate (834 mg,
7.87 mmol, 1.5 eq), tetrabutylammonium chloride (1.46 g, 5.25 mmol)
and copper(I) iodide (1.00 g, 5.25 mmol, 1 eq). The mixture was
stirred at room temperature for 48 h. Ether (30 ml) and 1M HCl (30
ml) were then added. After filtration through a bed of celite, the
organic phase was washed with brine, dried over sodium sulfate and
the solvent was evaporated under reduced pressure. Purification of
the residue by flash column chromatography on silica gel with
ether-hexane (40:60) as the eluent gave the product 0.103 (442 mg,
62%) as a colourless oil. Found: C, 79.55; H. 8.82. Calc. for
C.sub.9H.sub.12O: C, 79.37; H, 8.88%. .nu..sub.max (film)/cm.sup.-1
3650-3100 (br), 2975 (s), 2938 (s), 2905 (s), 2880 (s), 2500 (m),
1415 (m), 1375 (w), 1320 (s), 1180 (w), 1120 (w), 1040 (s), 900
(m), 735 (w); .delta..sub.H (300 MHz, CDCl.sub.3) 1.10 (3H, t, J
7.4, C9-H.sub.3), 1.96 (H, bs, OH), 2.13-2.20 (2H, m, C8-H.sub.2),
2.41-2.45 (2H, m, C2-H.sub.2), 3.11-3.13 (2H, m, C5-H.sub.2), 3.69
(2H, t, J 6.1, C1-H.sub.2); .delta..sub.C (Acetone, 300 MHz) 10.14,
13.07, 14.72, 24.03, 61.95, 75.08, 76.83, 78.46, 82.42; m/e (EI):
135 [(M-H).sup.+, 12%], 121 (44), 107 (30), 105 (51), 103 (29), 93
(44), 91 (100), 79 (58), 77 (80); 65 (41), 63 (29), 57 (14), 53
(27), 51 (37); HRMS: found m/e 135.081144 (M-H).sup.+; calc. for
C9H.sub.11O: 135.080990.
[0280] (3Z,6Z)-Nona-3,6-dien-1-ol, 104. Nona-3,6-diyn-1-ol 103 (198
mg, 1.45 mmol) was hydrogenated at atmospheric pressure, in the
presence of a mixture of quinoline (44 mg) and palladium (5%) on
calcium carbonate (100 mg), poisoned with lead in methanol (25 ml).
The reaction was stopped after 2.5 h when the uptake of hydrogen
was 61 ml. Removal of methanol in vacuo, followed by silica gel
column chromatography to remove quinoline using ether-hexane
(35:65) as the eluent gave 187 mg (92%) of
(3Z,6Z)-nona-3,6-dien-1-ol 104 as a colourless oil. Found: C,
77.42; H, 11.75. Calc. for C.sub.9H.sub.16O: C, 77.09; H, 11.50%.
.nu..sub.max (film)/cm.sup.-1 3500-3160 (br), 3011 (s), 2960 (s),
2930 (s), 2870 (s), 1462 (m), 1377 (m), 1050 (m), 722 (m); 8H (300
MHz, CDCl.sub.3) 0.97 (3H, t, J 7.6, H9-H.sub.3), 2.01-2.12 (2H, m,
C8-H), 2.32-2.40 (2H, m, C2-H.sub.2), 2.79-2.84 (2H, t, J 7.1,
C5-H.sub.2), 3.64 (2H, m, C1-H.sub.2), 5.27-5.43 (3H, m), 5.49-5.56
(H, m); SC (300 MHz, CDCl.sub.3) 14.82, 21.14, 26.20, 31.33, 62.77,
125.90, 127.40, 132.04, 132.74; m/e (EI): 140 (M.sup.+, 2%); 122
(15), 111 (7), 109 (12), 107 (22), 98 (12), 96 (19), 95 (21), 93
(72), 91 (33), 81 (39), 79 (56), 68 (31), 67 (100), 55 (59), 54
(21), 53 (21); HRMS: found m/e 140.120290 (M.sup.+); calc. for
C.sub.9H.sub.16O: 140.120115.
[0281] (3Z,6Z)-Nona-3,6-dienyl p-toluenesulfonate, 105.
(3Z,6Z)-Nona-3,6-dien-1-ol 104 (167 mg, 1.19 mmol) was dissolved in
chloroform (5 ml) and the solution was cooled in an ice bath.
Pyridine (376 mg, 4.76 mmol, 4 eq) was then added, followed by the
addition of p-toluenesulfonyl chloride (340 mg, 1.78 mmol, 1.5 eq)
in small portions with constant stirring. The mixture was stirred
for 24 h at 15.degree. C. Ether (15 ml) and water (5 ml) were added
and the organic layer was washed successively with 1 N HCl (10 ml),
5% NaHCO.sub.3, water (10 ml), and brine (10 ml), and then dried
over Na.sub.2SO.sub.4. The solvent was removed under reduced
pressure and the crude tosylate was flash column chromatographed on
silica gel with ether-hexane (20:80) as the eluent to yield
starting material (15 mg, 9%) and the title product 105 (201 mg,
57%) as a colourless oil. Found: C, 65.17; H, 7.44; S, 11.27. Calc.
for C.sub.16H.sub.22SO.sub.3: C, 65.28; H, 7.53; S, 10.89%.
.nu..sub.max (film)/cm.sup.-1 3005 (m), 2960 (s), 2930 (m), 2870
(m), 1599 (m), 1462 (m), 1377 (s), 1310 (w), 1290 (w), 1189 (s),
1178 (s), 1100 (s), 1020 (w), 973 (s), 813 (s), 770 (m), 660 (s);
.delta..sub.H (300 MHz, CDCl.sub.3) 0.95 (3H, t, J 7.6,
C9-H.sub.3), 2.00-2.05 (2H, m, C8-H.sub.2), 2.38-2.44 (2H, m,
C2-H.sub.2), 2.45 (3H, s, ArCH.sub.3), 2.69-2.74 (2H, t, J 7.0,
C3-H.sub.2), 3.994.04 (2H, m, C1-H.sub.2), 3.20-5.28 (2H, m),
5.345.50 (2H, m) 7.33, 7.80 (4H, dd, J 8.2 and 8.7, AA'BB' and
ArH); .delta..sub.C (300 MHz, CDCl.sub.3) 14.78, 21.09, 22.20,
26.12, 27.64, 70.20, 123.53, 126.94, 128.47, 130.37, 132.61,
132.92, 145.28; m/e (EI): [277 (M-OH).sup.+, 1%], 155 (25), 139
(2), 122 (67), 107 (47), 93 (100), 91 (77), 79 (66), 67 (47), 55
(32); m/e (CI): 312 (M+NH.sub.4).sup.+.
[0282] 3-[(3Z,6Z)-Nona-3,6-dienylthio]propionic acid, 106.
3-Mercaptopropionic acid (150 mg, 1.41 mmol, 1.5 eq) was added,
under an atmosphere of dry nitrogen, to a stirred solution of
sodium methoxide, prepared from sodium (64 mg, 2.78 mmol, 3 eq) and
methanol (20 ml). After the initial white precipitate had
dissolved, a solution of (3Z,6Z)-nona-3,6-dienyl p-toluenesulfonate
105 (276 mg, 0.94 mmol) in diethyl ether was added. The mixture was
stirred at 40.degree. C. for 2 days under nitrogen, then
hydrochloric acid (10% v/v, 20 ml) and diethyl ether (20 ml) were
poured into the crude reaction mixture. The organic phase was
separated and washed with water and brine, and dried over sodium
sulfate. After removal of the solvent, the residue was purified by
flash column chromatography using ether-hexane-acetic acid
(60:40:2) as the eluent to afford
3-[(3Z,6Z)-noca-3,6-dienylthio]propionic acid 106 (88 mg, 41%) as a
colourless oil. Found: C, 62.90; H, 8.73; S, 14.01. Calc. for
C.sub.12H.sub.20SO.sub.2: C, 63.12; H, 8.83; S, 14.04%.
.nu..sub.max (film)/cm.sup.-1 3400-2500 (br), 3005 (m), 2960 (m),
2910 (m), 2870 (w), 1713 (s), 1459 (m), 1377 (w), 1264 (m), 1195
(w), 1140 (w), 940 (w); .delta..sub.H (500 MHz, CDCl.sub.3) 0.97
(3H, t, J 7.8, C9'-H.sub.3), 2.05-2.08 (2H, m, C8'-H.sub.2),
2.34-2.39 (2H, m, C2'-H.sub.2), 2.57-2.60 (2H, t, J 7.4,
C1'-H.sub.2), 2.65-2.69 (2H, t, J 7.3, C3-H.sub.2), 2.78-2.82 (4H,
m, C5'-H.sub.2, C2-H.sub.2), 5.27-5.32 (H, m), 5.37-5.47 (3H, m),
5.50-6.10 (H, bs, COOH); .delta..sub.C (300 MHz, CDCl.sub.3) 14.83,
21.14, 26.20, 27.19, 27.95, 32.62, 35.21, 127.37, 127.97, 130.53,
132.72, 178.66; m/e (EI): 228 (M.sup.+, 34%), 169 (14), 159 (18),
155 (45), 133 (8), 122 (54), 119 (42), 113 (12), 107 (44), 93
(100), 89 (66), 79 (57), 77 (53), 67 (52), 61 (33), 55 (43); HRMS:
found m/e 228.118179 (M.sup.+); calc. for C.sub.12H.sub.20SO.sub.2:
228.118402.
[0283] 3-Tetradecylthiopropionic acid, 108. According to the
procedure described for the preparation of
3-[3Z,6Z)-nona-3,6-dienylthio]propionic acid 106,
3-mercaptopropionic acid (261 mg, 2.46 mmol, 1.2 eq) was added,
under an atmosphere of dry nitrogen, to a stirred solution of
sodium methoxide prepared from sodium (142 mg, 6.17 mmol, 3 eq) and
methanol (20 ml). After the initial white precipitate had
dissolved, a solution of 1-bromotetradecane 107 (568 mg, 2.05 mmol)
in diethyl ether (2 ml) was added. The reaction mixture was stirred
for 16 h at room temperature. After workup and purification by
flash column chromatography using ether-hexane (20:80)
ether-hexane-acetic acid (60:40:1) for elution, the title compound
108 (450 mg, 73%) was obtained as a white solid, mp: 67.degree. C.
Found: C, 67.32; H, 11.32; S, 10.41. Calc. for
C.sub.17H.sub.34SO.sub.2: C, 67.50; H, 11.33; S, 10.60%.
.nu..sub.max (Nujol)/cm.sup.-1 3100-2600 (br), 2965 (s), 2910 (s),
2840 (s), 1680 (s), 1460 (s), 1405 (w), 1375 (m), 1265 (m), 1255
(w), 1231 (w), 1210 (w), 1200 (m), 1080 (w), 915 (m), 725 (m);
.quadrature..sub.H (500 MHz, CDCl.sub.3) 0.88 (3H, t, J 6.7,
C14'-H.sub.3), 1.25-1.38 [22H, m, (C3'-C.sub.13')-H.sub.2],
1.56-1.61 (2H, m, C2'-H.sub.2), 2.54 (2H, bs, C1'-H.sub.2),
2.65-2.68 (2H, t, J 6.6, C3-H.sub.2), 2.79 (2H, bs, C2-H.sub.2);
.delta..sub.C (300 MHz, CDCl.sub.3) 14.69, 23.26, 27.16, 29.44,
29.80, 29.93, 30.02, 30.10, 30.17, 30.23, 32.49, 32.78, 35.25,
178.50; m/e (EI): 302 (M.sup.+, 21%), 230 (24), 229 (100), 185 (2),
161 (4), 119 (8), 106 (24), 97 (15), 89 (21), 83 (22), 69 (25), 55
(32); HRMS: found m/e 302.227166 (M.sup.+); calc. for
C.sub.17H.sub.34SO.sub.2: 302.27952.
[0284] 2-Tetradecylthioacetic acid, 109. 2-Mercaptoacetic acid (288
mg, 3.13 mmol, 1.2 eq) was added, under an atmosphere of dry
nitrogen, to a stirred solution of sodium methoxide, prepared from
sodium (180 mg, 7.83 mmol, 3 eq) and methanol (20 ml). After the
initial white precipitate had dissolved, a solution of
1-bromotetradecane 107 (725 mg, 2.61 mmol) in diethyl ether (2 ml)
was added and the mixture was stirred for 16 h at room temperature
under nitrogen. The crude reaction mixture was poured into an equal
volume of hydrochloric acid (10% v/v), and the organic phase was
separated and washed with water and brine, and dried over sodium
sulfate. After removal of the solvent, the residue was purified by
flash column chromatography using diethyl ether-hexane (20:80)
diethyl ether-hexane-acetic acid (60:40:2) for elution and
crystallised to afford 2-tetradecylthioacetic acid 109 (580 mg,
77%) as a white solid, mp: 68.degree. C. Found: C, 66.46; H, 10.93;
S, 10.83. Calc. for C.sub.16H.sub.32SO.sub.2; C, 66.61; H. 11.18;
S, 11.11%. .nu..sub.max (Nujol)/cm.sup.-1 3200-2600 (br), 2950 (s),
2910 (s), 2840 (s), 1680 (s), 1460 (s), 1425 (w), 1375 (s), 1265
(m), 1140 (w), 908 (w), 725 (w); .delta..sub.H (300 MHz,
CDCl.sub.3) 0.88 (3H, t, J 6.6, C14'-H.sub.3), 1.26-1.40 [22H, m,
(C3'-C13')-H.sub.2], 1.56-1.64 (2H, m, C2'-H.sub.2), 2.64-2.69 (2H,
t, J 7.4, C1'-H.sub.2), 3.26 (2H s, C2-H.sub.2); .delta..sub.C (300
MHz, CDCl.sub.3) 14.68, 23.26, 29.30, 29.46, 29.75, 29.93, 30.06,
30.15, 30.22, 32.49, 33.36, 34.05, 177.57; m/e (EI): 288 (M.sup.+,
12%), 230 (21), 279 (100), 111(6), 97 (17), 83 (27), 69 (30), 55
(34); HRMS: found m/e 288.212125 (M.sup.+); calc. for
C.sub.16H.sub.32SO.sub.2: 288.212302.
[0285] Propyl (all-Z)-eicosa-5,8,11,14-tetraenyl sulfide 110. Using
the procedure described for the preparation of
3-tetradecylthiopropionic acid 108, propanethiol (26 mg, 0.34 mmol,
1.2 eq) was added, under an atmosphere of dry nitrogen, to a
stirred solution of sodium methoxide, prepared from sodium (20 mg,
0.87 mmol, 3 eq) and methanol (10 ml). After the initial white
precipitate had dissolved, a solution of
(all-Z)-1-bromo-5,8,11,14-eicosatetrane 58c (101 mg, 0.29 mmol) in
diethyl ether (1 ml) was added. The reaction mixture was stirred
for 15 h at room temperature. After workup, purification by flash
column chromatography using hexane for elution gave the title
compound 110 (75 mg, 75%) as a colourless oil. Found: C, 78.91; H,
11.38; S, 8.96. Calc. for C.sub.23H.sub.40S: C, 79.24; H, 11.56; S,
9.20%. .nu..sub.max (film)/cm.sup.-1 3005 (s), 2950 (s), 2920 (s),
2850 (S), 1650 (W), 1450 (m), 1390 (w), 1375 (w), 1290 (w), 1260
(w), 1230 (w), 910 (w), 720 (m); .sup.5H (CDCl.sub.3, 300 MHz) 0.89
(3H, t, 16.8, C20-H.sub.3), 0.99 (3H, t, J 7.2, C3'-H.sub.3),
1.261.35 (6H, m, C17-H.sub.2, C18-H.sub.2, C19-H2), 1.43-1.48 (2H,
C3H2), 1.57-1.64 (4H, m, C2-H2, C2'-H2), 2.05-2.13 (4H, n, C4-H2,
C16-H2), 2.50-2.51 (4H, m, C1-H2, C1'-H2), 2.80-2.86 (6H, m, C7-H2,
C10-H2, C13-H2), 5.32-5.43 (8H, m, C5-H, C6-H, C8-H, C9-H, C11-H,
C12-H, C14-H, C15-H); 5C (CDCl.sub.3, 300 MHz) 14.13, 14.67, 23.17,
23.60, 26.22, 27.41, 27.81, 29.44, 29.91, 32.11, 32.54, 34.79,
128.12, 128.48, 128.64(2C), 128.90, 129.11, 130.40, 131.06; m/e
(EI): 348 (M+, 44%), 305 (38), 273 (4), 251 (6), 237 (14), 205
(17), 177 (19), 161 (36), 150 (27), 131 (29), 119 (40), 105 (48),
93 (77), 91 (76), 81 (79), 79 (95), 67 (100), 53 (69); HRMS: found
m/e 348.285378 (M.sup.+); calc. for C.sub.23H.sub.40S:
348.285073.
[0286] Propyl tetradecyl sulfide, 111. Using the procedure
described above for the synthesis of propyl
(all-Z)-eicosa-5,8,11,14-tetraenyl sulfide 110, propanethiol (165
mg, 2.16 mmol, 1.2 eq) was added, under an atmosphere of dry
nitrogen, to a stirred solution of sodium methoxide, prepared from
sodium (82 mg, 3.56 mmol, 2 eq) and methanol (10 ml). After the
initial white precipitate had dissolved, a solution of
1-bromotetradecane 107 (500 mg, 1.80 mmol) in diethyl ether (2 ml)
was added. The reaction mixture was stirred for 15 h at room
temperature. After workup, purification by flash column
chromatography using hexane for elution gave the title compound 111
(435 mg, 89%) as a colourless oil. Found: C, 75.05; H, 13.27; S,
11.50. Calc. for C.sub.17H.sub.36S: C, 74.92; H, 13.31; S, 11.76%.
.nu..sub.max (film)/cm.sup.-1 2960 (s), 2910 (s), 2850 (s), 1460
(s), 1375 (w), 1290 (w), 1270 (w), 890 (w), 720 (w); .delta..sub.H
(CDCl.sub.3, 300 MHz) 0.87 (3H, t, J 6.5, C14-H3), 0.99 (3H, t, J
7.4, C3'-H.sub.3), 1.25 [22H, m, (C3-C13)-H.sub.2], 1.54-1.63 (4H,
m, C2-H.sub.2, C2'-H.sub.2), 2.47-2.51 (4H, m, C1-H.sub.2,
C1'-H.sub.2); .delta..sub.C (CDCl.sub.3, 300 MHz) 14.13, 14.71,
23.28, 23.59, 29.55, 29.85, 29.94, 30.12, 30.18, 30.23, 30.33,
32.50, 32.69, 34.78; m/e (EI): 272 (M.sup.+, 52%), 243 (18), 229
(100), 196 (8), 187 (2), 168 (5), 145 (6), 131 (15), 111 (14), 97
(2), 89 (34), 83 (27), 76 (33), 69 (32), 57 (30), 55 (44).
[0287] 3-(Tetradecylsulfinyl)propionic acid, 113. Arachidonic acid
1 (175 mg) was dissolved in 5 ml of dichloromethane to make a stock
solution (35 mg/ml). 3-Tetradecylthiopropanoic acid 108 (10 mg,
0.03 mmol), arachidonic acid 1 (10 mg, 0.03 mmol, 284 .mu.l) and
dichloromethane (10 ml) were added into a one-neck flask (500 ml).
The solvent was evaporated using a rotary evaporator to allow the
reagents to form a thin film on the internal surface of the flask.
The flask was filled with oxygen and placed in darkness for 7 days.
Dichloromethane (5 ml) was then added into the flask to dissolve
the mixture and the solution was then transferred to a 2 ml vial.
After evaporation of the solvent, the residue was dissolved in 300
.mu.l of the mobile phase (methanol-30 mM phosphoric acid, 90:10)
and then subject to reverse phase HPLC analysis. The HPLC was
performed on an Alltech Spherisorb octadecylsilane (ODS) column
with RI detection. The flow rate of the mobile phase was 3 ml/min.
Fifty microlitres of the sample was loaded each time. The product
with a retention time of 5.49 min was collected and pooled. After
evaporation of the solvent at reduced pressure, the product was
extracted with diethyl ether (2 ml). The resulting extract was
washed with water and dried with Na.sub.2SO.sub.4 and the solvent
evaporated, yielding the title compound 113 (2 mg) as a white
solid, mp: 166-167.degree. C. Found: 64.33, H, 10.50. Calc. for
C.sub.17H.sub.34SO.sub.3: C, 64.11; H, 10.76%. .nu..sub.max
(Nujol)/cm.sup.-1 3600-2500 (br), 2965 (s), 2910 (s), 2840 (s),
1695 (m), 1460 (s), 1375 (s), 1330 (w), 1305 (w), 1125 (w), 1040
(w), 1025 (w), 920 (w), 720 (w); .delta..sub.H (CDCl.sub.3, 500
MHz) 0.81 (3H, t, J 7.0, C14'-H.sub.3), 1.19-1.26 [20H, m,
C4'-C13')-H.sub.2], 1.34-1.37 (2H, m, C3'-H.sub.2), 1.68-1.72 (2H,
m, C2'-H.sub.2), 2.70-2.76 (H, m), 2.82-2.89 (3H, m), 2.88-3.03 (H,
m), 3.05-3.10 (H, m), 7.96 (H, bs, COOH); .delta..sub.C
(CDCl.sub.3, 300 MHz) 14.67, 23.19, 23.24, 27.78, 29.29, 29.72,
29.91, 30.09, 30.17, 30.20, 32.47, 46.66, 52.53, 174.37; m/e (CI):
319 (MH.sup.+); m/e (EI): 301 [(M-OH).sup.+, 27%], 246 (21), 245
(16), 279 (100), 196 (5), 121 (15), 94 (22), 97 (22), 83 (29), 71
(32), 70 (34), 57 (51); HRMS: found m/e 301.219714 (M-OH).sup.+;
calc. for C.sub.17H.sub.33SO.sub.2: 301.220127.
[0288] 2-(Tetradecylsulfinyl)acetic acid, 114.
2-Tetradecylthioacetic acid 109 (19 mg, 0.066 mmol) was dissolved
in dichloromethane (2 ml) and tert-butylhydroperoxide (11 ml, 0.08
mmol, 1.2 eq) was added. After 48 h reaction at room temperature,
the solvent was removed and the residue was chromatographed using
ether-hexane-acetic acid (60:40:2).fwdarw.methanol as the eluent to
obtain the white product 114 (17 mg, 86%). .delta..sub.H
(CDCl.sub.3, 300 MHz) 0.88 (3H, t, J 6.4, C14'-H.sub.3), 1.20-1.29
[20H, m, (C4'-C13)-H.sub.2], 1,44-1.50 (2H, m, C3'-H.sub.2),
1.77-1.82 (2H, m, C2'-H.sub.2), 2.88-2.95 (H, m, C1'-H), 3.02-3.07
(H, m, C1'-H'), 3.63-3.68 (H, d, J 14, C2-H), 3.81-3.86 (H, d, J
14, C2-H'), 7.92 (H, bs, COOH); .delta..sub.C (CDCl.sub.3, 300 MHz)
14.69, 23.20, 23.26, 29.18, 29.70, 29.89, 29.93, 30.09, 30.18,
30.22, 32.49, 52.27, 53.47, 166.93; m/e (EI): 305 [(M+1).sup.+,
1%], 287 (50), 243 (60), 229 (94), 196 (12), 168 (6), 149 (6), 125
(10), 111 (21), 97 (45), 83 (63), 69 (74), 57 (100), 53 (91); HRMS:
found m/e 305.215275 (M+1).sup.+ calc. for
C.sub.16H.sub.33SO.sub.3: 305.215042.
CONCLUSION
[0289] The main group of compounds targeted in this project was the
nitro analogues of PUFAs. They were expected to be potentially
useful due to their generally high stability and the chemical
similarity of the nitro group to the carboxyl group.
[0290] From the nine nitro analogues of PUFAs that were
synthesised, including long chain nitroalkanes, y-nitro fatty acids
and carboxyethyl nitro fatty acids,
(all-Z)-4-nitrotricosa-8,11,14,17-tetraenoic acid has been
identified as a good substrate of soybean 1-LO and a 12-LO from
porcine leukocytes. The substrate activity of this compound with
the soybean 15-LO is comparable to that of arachidonic acid, which
is a major substrate of the lipoxygenase.
[0291] A more significant outcome of this work was the
identification of 4-nitrohenicosanoic acid,
3-(all-Z)-nonadeca-4,7,10,13-tetraenyl]-3-nitro-
pentane-1,5-dicarboxylic acid and
3-heptadecyl-3-nitropentane-1,5-dicarbox- ylic acid as selective
inhibitors of 5-LO, 12-LO and 15-LO catalysed oxidation of
arachidonic acid, respectively. Although a large number of
inhibitors have been reported for these three lipoxygenases, so far
few inhibitors have entered clinical trials and no agents that are
selective for 15-LO vs 5-LO (or vs 12-LO) are
available..sup.[65]
[0292] Selective inhibition of a specific lipoxygenase is
particularly desirable for treatment of diseases related to these
metabolic pathways. Non-selective inhibitors have the disadvantages
of causing possible side effects. For instance, asthma has been
treated as an inflammatory disease, and corticosteroids are the
therapy of choice for the inflammatory component of
asthma..sup.[66] Although this class of drugs provides powerful
anti-inflammatory effects in most patients, these effects are not
specific and in some cases result in serious side effects. Since
leukotrienes, a family of inflammatory mediators generated through
the 5-LO pathway, have been shown to enhance bronchoconstriction
and airway mucus secretion, agents that target the specific
inflammatory pathway have been developed to treat asthma by
modulating leukotriene activity. So far, specific leukotriene
receptor antagonists and synthesis inhibitors have been extensively
studied in laboratory-induced asthma and currently show promise in
clinical trials; one leukotriene receptor antagonist (zafirlukast)
and one 5-LO inhibitor (zileuton) were recently approved for the
treatment of asthma..sup.[66] The identification of the three nitro
analogues of PUFAs having selective inhibition activity with the
three lipoxygenases may lead toward a new class of drugs with
specificity and reduced side effects for treating diseases that are
associated with lipoxygenase pathways.
[0293] It will be appreciated by persons skilled in the art that
numerous variations and/or modifications may be made to the
invention as shown in the specific embodiments without departing
from the spirit or scope of the invention as broadly described. The
present embodiments are, therefore, to be considered in all
respects as illustrative and not restrictive.
REFERENCES
[0294] 1. Ferrante, A., Hii, C. S. T., Huang, Z. H., Rathjen, D. A.
In The Neutrophils: New Outlook for the Old Cells. (Ed.
Gabrilovich, D.) Imperial College Press (1999) 4: 79-150.
[0295] 2. Sinclair, A., and Gibson, R. (eds) 1992. Invited papers
from the Third International Congress. American Oil Chemists'
Society, Champaign, Ill. 1-482.
[0296] 3. Abel, S., Gelderblom, W. C. A., Smuts, C. M., Kruger M.
Pros. Leuko. and Essential, 56 (1): 29-39 (1997).
[0297] 4. Krombout, D. Nutr. Rev. 50:49-53 (1992).
[0298] 5. Kinsella, J. E., Lokesh, B., Stone R. A. Am. J. Clin.
Nutr. 52:1-28 (1990)
[0299] 6. Kumaratilake, L. M., Robinson, B. S., Ferrante, A.,
Poulos A. J. Am. Soc. Clin. Investigation 89: 961-967 (1992).
[0300] 7. Weber, P. C. Biochem. Soc. Trans. 18:1045-1049
(1990).
[0301] 8. Arm, J. P., and Lee, T. H. Clin. Sci. 84: 501-510
(1993).
[0302] 9. Thien, F. K. C. K., and Walters, E. H. Pros. Leuko and
Essential 52: 271-288 (1995).
[0303] 10. Ford-Hutchinson, A. W. Crit. Rev. Immunol. 10(1): 1
(1990).
[0304] 11. Bates, E. J. Pros. Leuko and Essential. 53: 75-86
(1995).
[0305] 12. Ferrante, A., Poulos, A., Easton, C. J., Pitt, M. J.,
Robertson, T. A., Rathjen, D. A. International Patent Application
No. PCT/AU95/00677 (1995)-WO96/11908: Chem. Abstr. 125:
58194-(1996).
[0306] 13. Pitt, M. J., Easton, C. J., Moody, C. J., Ferrante, A.,
Poulos, A., and Rathjen, D. A. Synthesis 11:1239-1242 (1997).
[0307] 14. Barnes, N. C., Hui, P. K. Pulmonary Pharmacol. 6(1): 39
(1993).
[0308] 15. Kornblum, N., Taub, B., Ungnade, H. E. J. Am. Chem. Soc.
76:3209-3211 (1954).
[0309] 16. Chasar, D. W. Synthesis 84182 (1982).
[0310] 17. Pollini, G. P., Barco, A., and de Guili, G. Synthesis.
44-45 (1972).
[0311] 18. Corey, E. J., and Suggs, J. W. Tetrahedron Letters
31:2647-2650 (1975).
[0312] 19. Rosini, G., Ballini, R., and Petini, M. Synthesis
269-271 (1985).
[0313] 20. Melton, J., and McMurry, J. E. J. Org. Chem. 4(14)
(1975).
[0314] 21. Finkbeiner, H. L., and Wagner, G. W. J. Org. Chem. 28:
215 (1963).
[0315] 22. Finkbeiner, H. L., and Stiles, M. J. Am. Chem. Soc. 85:
616-632 (1962).
[0316] 23. Hayashi, H.; Nakanishi, K.; Brandon, C.; Marmur, J. J.
Am. Chem. Soc. 1973, 95, 8749.
[0317] 24. Kornblum, N.; Taub, B.; Ungnade, H. E. J. Am. Chem. Soc.
1954, 76, 3209.
[0318] 25. a) Stiles, M.; Finkbeiner, H. L. J. Am. Chem. Soc. 1959,
81, 505.
[0319] b) Finkbeiner, H. L.; Wagner, G. W. J. Org. Chem. 1963, 28,
215.
[0320] 26. Seebach, D.; Lehr, F. Angew. Chem., Int. Ed. Engl. 1976,
15, 505.
[0321] 27. a) Finkbeiner, H. L.; Stiles, M. J. Am. Chem. Soc. 1963,
85, 616.
[0322] b) Feuer, H.; Hass, H. B.; Warren, K. S. J. Am. Chem. Soc.
1949, 71, 3078.
[0323] 28. Chasar, D. W. Synthesis 1982, 841.
[0324] 29. Baldwin, J. E.; Au, A.; Christie, M.; Haber, S. B.;
Hesson, D. J. Am. Chem. Soc. 1975, 97, 5957.
[0325] 30. Corey, E. J.; Suggs, J. W. Tetrahedron Lett. 1975, 31,
2647.
[0326] 31. Rosini, G.; Ballini, R. Synthesis 1988, 833.
[0327] 32. Ballini, R.; Bosica, G.; Forconi. P. Tetrahedron 1996,
52, 1677.
[0328] 33. Melton, J.; McMurry, J. E. J. Org. Chem. 1975, 40,
2138.
[0329] 34. a) Porter, N. A.; Wolf, R. A.; Yarbro, E. M.; Weenen, H.
Biochem. Biophys. Res. Commun. 1979, 89, 1058.
[0330] b) Porter, N. A.; Logan, J.; Kontoyiannidou, V. J. Org.
Chem. 1979, 44, 3177.
[0331] c) Terao, J.; Matsushita, S. Agric. Biol. Chem. 1981, 45,
587.
[0332] 35. Corey, E. J., and Park, H. J. Am Chem. Soc. 104:
1750-1752 (1982).
[0333] 36. NCI Press Office 1997, Recent trends in prostate cancer
incidence and mortality. National Cancer Institute.
[0334] 37. Petrylak 1999 Urology 54: 30-35
[0335] 38. Cleeve 1999 Prostate News, Prostate Cancer Society of
Austalia, 3
[0336] 39. Greenberg et al 1995 PNAS 92: 3439-3443
[0337] 40. Chen et al 1992 Steroids, 57:269-275
[0338] 41. Zhao et al 1997 J Biol Chem, 272.22751-22757
[0339] 42. Bartsch et al 1999 Carcinogenesis 20:2209-2218
[0340] 43. Yang et al 1999 Clin Biochem, 32:405-409
[0341] 44. Norrish et al 1999 Br J Cancer 81:1238-1242
[0342] 43. Hii et al 1995 Carcinogenesis 16:1505-1511
[0343] 46. Couper et al 1998 Human Immunology 59:493-499
[0344] 47. Hii et al 1995 J Biol Chem 270: 4201-4204
[0345] 48. Hardy et al 1995 Biochem J, 311: 689-697
[0346] 49. Huang et al 1997 Biochem J. 325: 553-557
[0347] 50. Hii et al 1999 in Eicosanoids and Other Bioactive Lipids
in Cancer, Inflammation and Related Diseases (Honn, K. V., Nigam,
S., Marnett, L. J. and Dennis, E. eds), pp 365-370, Plenum Press,
NY.
[0348] 51. Ferrante et al 1999 in The Neutrophils: New Outlook for
the Old Cells (Gabrilovich, D. ed) 4: 79-150, Imperial College
Press.
[0349] 52. Anderson et al 1998 Prostate, 37: 161-173
[0350] 53. Ghosh and Myers 1998 PNAS, 95: 13182-13187
[0351] 54. Palayeor et al 1999 Oncogene, 18: 7389-7394
[0352] 55. Ford-Hutchinson 1991 Trends Pharmacol Sci, 12: 68-70
[0353] 56. Avis et al 1996 J. Clin Invest., 97: 806-813
[0354] 57. Australian Institute of Health and Welfare (AIHW).
Breast and Cervical Cancer Screening in Australia 1996-97. 1998.
Canberra, Australian Institute of Health and Welfare.
[0355] 58. Johnson-Thompson M. C. and Guthrie, J. (2000) Cancer,
88, 1224-1229.
[0356] 59. Bartsch, H., Nair, J. and Owen, R. W. (1999)
Carcinogenesis, 20, 22209-2218.
[0357] 60. Rose, D. P. and Connolly, J. M. (1997) Breast Cancer Res
Treat, 46, 225-237.
[0358] 61. Steele, V. E., Holmes, C. A., Hawk, E. T., Kopelovich,
L., Lubet, R. A., Crowell, J. A., Sigman, C. C. and Kelloff, G. J.
(1999) Cancer Epidemiol Biomarkers Prev, 8, 467-483.
[0359] 62. Przylipiak, A., Hafner, J., Przylipiak, J., Kohn, F. M.,
Runnebaum, B. and Rabe, T. (1998) Gynecol Obstet Invest, 46,
61-64.
[0360] 63. Connolly, J. M. and Rose, D. P. (1998) Cancer Lett, 132,
107-112.
[0361] 64. Natarajan, R., Esworthy, R., Bai, W., Gu, J. L.,
Wilczynski, S. and Nadler, J. (1997) J Clin Endocrinol Metab, 82,
1790-1798.
[0362] 65. Editorial, J. Clin. Invest. 99: 1147-1148 (1997).
[0363] 66. Wenzel, S. E. Am. J. Med. 104:287-300 (1998).
[0364] 67. Pitt M J., Easton C J. Ferrante A., Poulos, A., Rathjen
D A. Chem. Phys. Lipids 92: 63-69 (1998).
* * * * *