Note: Descriptions are shown in the official language in which they were submitted.
CA 02340484 2001-03-19
-1-
Background of the Invention
Field of the Invention
This invention relates to a novel chemical synthesis
of stable, water-soluble chemiluminescent 1,2-dioxetanes
and to novel intermediates obtained in the course of
synthesizing such 1,2-dioxetanes.
This Application is a Divisional of Canadian Patent
Application S.N. 2,035,029, filed September 4, 1990.
Description of Related Art
1,2-Dioxetanes, cyclic organic peroxides whose
central structure is a four-menibered ring containing
pairs of contiguous carbon and oxygen atoms (the latter
forming a peroxide linkage), are a known, but until
recently seldom utilized, class of compounds. Some 1,2-
dioxetanes can be made to exhibit chemiluminescent
decomposition, e.g., by the action of enzymes, as
described in the following: Bronstein, PCT WO 88/00695,
published January 28, 1988, "Method of Detecting a
Substance Using Enzymatically-Induced Decomposition of
Dioxetanes"; Bronstein et al, PCT WO 89/06650, published
July 27, 1989, "Dioxetanes for Use in Assays"; Edwards,
PCT WO 89/06226, published July 13, 1989, "Synthesis of
1,2-Dioxetanes and Intermediates Therefor"; Edwards _et
al, U..S. Patent 4,952,707, issued August 28, 1990, "Novel
Chemiluminescent Fused Polycyclic Ring-Containing 1,2
Dioxetanes and Assays in Which They are Used", as well as
in Bronstein, I.Y. et al, "Novel Enzyme Substrates and
Their Application in Immunoassay", J. Biolum. Chem. 2:186
(1988) .
The amount of light emitted during such chemilumi-
nescence is a measure of the concentration of a
.. .. . _..._ ~_.... _._. "...r._.~_;~.-02340484 2001-03-19 -'~"' -
..>_......_.~ .._.,......... _..._, .~..,._.. ._..._ _. .
2
luminESCent substance which, in turn, is a measure of
the concentration of its precursor 1,2-dioxetane. Thus,
by measuring the intensity of luminescence, the
concentration of the 1,2-dioxetane, and hence the
concentration of a substance being assayed (e.g:, a
biological species bound to the 1,2-dioxetane member of
a specific binding pair in a bioassay) can be
determined. The appropriate choice of substituents on
the 1,2-dioxetane ring allows, her alia, for adjustment
l0 of the chemical stability of the molecule which, in
. turn, affords a means of controlling the onset of
chemiluminescence, thereby enhancing the usefulness of
such chemiluminescence for practical purposes, e.g.,
immunoassays, nucleic acid probe assays, enzyme assays,
and the like.
The preparation of 1,2-dioxetanes by
photo-oxidation of olefinic double bonds is known.
Mazur, S. stet al., ~. Am. Chem. Soc., 92:3225 (1970).
However a need exists for a facile, general synthesis of
substituted 1,2-dioxetanes from olefinically-unsaturated
precursors derived from readily available or obtainable
starting materials through tractable intenaediates. In
this connection, a particular need exists for a
commercially useful method for producing 1,2-dioxetanes
of the general formula:
OR3
Y-Z !I)
wherein T, R3, Y and Z are defined herein below, from
enol ether-type precursors of the general formula:
T ~ OR3
~ Y-Z
!II)
~ 02340484 2001-03-19 . ........... ... . .. . ......
' 3
McMurry ~ ~ (McMurry, J.E., g~ ~, J. Org.
Chem., 43:3255 (1978j] described titanium-induced .
reductive coupling of carbonyl groups to form olefins.
Schaap, A.P., EPO 254,051, published January 27; 1988,
and Bronstein, I.Y., 1986, disclose the use of this
reaction to produce compounds of formula (IIj by the
following general reaction:
_ O
i ~ ~ ~1~ ~ oR3
T~, O + R O Y ~ Z IriAl~ T~
Y- Z
Several problems with aforementioned unsymmetrical
McMurry coupling are especially important in the radical
based mechanism which operates in the above equation
when compared with similar mixed couplings between
aliphatic and dinryl ketoses where the mechanism is
ionic in nature. The need to often use molar excesses
of the expensive T = O ketone over ester co-reactants in
an attempt to favor the mixed coupling product, while at
the same time obtaining low yields at best, makes this
approach suitable only !or small scale preparations.
Fl~rthermore,~ the well-known capricious nature of the
reaction, the large amounts of TiCls/LiAlH; required to
effect the coupling, and the tormation of by-products
which are difficult to sepazate from the desired enol
ethers also limit the commercial utility o! the process.
In addition, certain useful mete-substituted starting
materials such as:
0 0~ o o~ o ~ oR3
~ ~ ..
a . ar ,
o-c-c ta~3~ j '' ~c . . ' s-
CA 02340484 2001-03-19 ., . .. .........._.~...,. _".....,..,~....:.~o-!-==
..~....,."_ ~: i..
4
cannot be used with the McMurry reagents as such substi-
tuent groups would be reduced, hydrolysed, or would take
part in reductive coupling with T ~ O. Thus,. the double
bond cannot be introduced regiospecifically in every
case.
Enol ethers have also been prepared by Peterson or
Wittig reactions of alkoxymethylenesilanes or
phosphoranes with aldehydes or ketones in basic media
[Magnus, P., g~ ~, Organometallics, 1:553 (1982):
Wynberg, H. and Meijer, ~.W., Tetrahedron Lett., 41:3997
(1979)]. Bronstein, 1986, above, describes the
synthesis of an olefin of formula (II) above using a
Wittig reaction of a phosphonium glide with a T = o
ketone. A major advantage of the Wittig reaction is
that it is an ionic reaction, where the double bond can
be introduced regiospecifically in almost every case.
One problem with the Wittig reaction, however, is
that the product alkene is difficult to separate from
the phosphine oxide by-product because of the similar
solubility characteristics of these compounds. Another
problem. is that the initially-produced phosphonium
glides can be made only from relatively expensive
phosphine starting materials [Walker, B.J., in Cadog~an.
J.I.G.. ed.. ~'Orcanophosohorus Reagents in Or aq nic
Synthesis" Academic Press. N.Y., (1978), pp. 155-205].
Also, as phosphonium glides are relatively weakly
nucleophilic, they will react only with a limited range
of carbonyl compounds, and can require relatively harsh
reaction conditions to do this [Gushurst, A.J., gt ~,
J. Ora. Chem., 53:3397 (1988)]. Finally, side reactions
frequently occur in the Wittig reactions, jahich also
contribute to relatively low yields [Hornet, L., g~ ~,
Chem. Her., 95:581 (1962)]:
Because of the many problems attendant upon both
the McMurry and Wittig reactions, particularly when used
_ . ~ 02340484 2001-03-19 1.n......_s.J~".~",J..:...,..;_...>,__
:.,....,.".;:.. . . . . ... ..
to synthesize olefinic intermediates for enzyme-
cleavable 1,2-dioxetanes on a commercial scale, a
more-suitable route to enol ether derivatives useful in
the synthesis of stable, water-soluble, enzyme-cleavable
5 chemiluminescent 1,2-dioxetanes was needed.
Summary of the Invention
This invention fills this need. A new synthesis of
stable, water-soluble chemiluminescent 1,2-dioxetanes,
particularly ones that are enzyme-cleavable, substituted
with stabilizing and solubilizing groups and ring-
containing fluorophore moieties, that avoids problems
inherent in previously-employed reactions, has now been
discovered. In particular, this invention is concerned
with a synthetic route to such 1,2-dioxetanes that
employs, for the first time, dialkyl 1-alkoxy-1-
arylmethane phosphonate-stabilized carbanion
intermediates in the synthesis of key enol ether
intermediates for the desired 1,2-dioxetane end
products.
The use of phosphonate-stabilized carbanions in a
Horner-Emmons reaction [Homer, L., g~ ~, Chem.'Ber.,
91:61 (1958): Wadsworth, W.S., J. Am. Chem. Soc.,
83:1733 (1961)] for the production of enol ethers used
in the synthesis of stable, water-soluble,
chemiluminescent 1,2-dioxetanes such as those of formula
(I) above, has been found to exhibit several advantages
over previous methods for synthesizing such enol ether
intermediates. These include: regiospecific
introduction of the olefinic double bond in the presence
of a wide range of ancillary functional groups;
increased nucleophilicity compared to the phosphonium
glides, which not only increases the variety of ketoses
with which the phosphonate-stabilized carbanions can ,_
react, but also permits this reaction to be carried out
under milder conditions; more-readily separable alkene
and phosphorous-containing byproducts than can be
,9
.. ..._. ..._.:~ 02340484 2001-03-19 . .. _..........._ ........ ... .._._:_
..... . . . ......
6
obtained using the Wittig reaction (the phosphoric acid
diester salt by-products produced by practicing this
invention are highly water-soluble): facile betaine
formation due to enhanced reactivity and stability of
the phosphonate carbanions compared to the phosphonium
ylides; and starting materials, i.e., trialkylphos-
phites, that are more cheaply and conveniently prepared
than the more-expensive phosphines necessary for the
Wittig reaction.
It has also been discovered that, not only does the
reaction of an arylaldehyde dialkyl acetal with a
trialkylphosphite or a trialkylsilyldialkylphosphite in
the presence of a Lewis acid [Bunkhouse, D.,
~,vnthesis, 330 (1984): Oh, D.Y., g~ ,~, Syn. Comm.,
16(8) 859 (1986)] provide a general and facile route to
the phosphonate intermediates for Horner-Emmons
reactions with T = 0 ketones or diones (O = T = O) than
does the previously known route employing the Arbuzov
reaction [Arbuzov, A.E., gt ~, them. Ben., 60:291
(1927)] between an alpha alkoxy arylmethyl halide and a
trialkylphosphite, but also that the aryl moiety of the
thus-employed arylaldehyde dialkyl acetal, which may be
open chain or cyclic (e. g., a 1,3-dioxolane or dioxane),
can be substituted with electron-donating or
-withdrawing meta-substituents. The resulting
~neta-substituted dialkyl 1-alkoxy-1-arylmethane
phosphonates, with one exception not useful in the
present invention [Creary, X., g~ ~,, J. Ora. Chem.,
50:2165 (1985)], are unknown in the prior art.
Meta-substituted aryl gzoups are preferred, as the
ultimate production of an electrondonating moiety in
this position, relative to the point of attachment of a
1,2-dioxetane group, has been found to maximize the
efficiencies for production of singlet excited states ..
from 1,2-dioxetanes such as those of formula (I) above,
substituted at the 4-position of the dioxetane ring with
CA 02340484 2001-03-19
-
a monocyclic or polycyclic aromatic ring-containing,
fluorophore-forming group.
And, as disclosed and claimed in copending Edwards
et al, U.S. Patent 4,952,707, when fused polycyclic
aromatic ring-containing, substituted dialkyl 1-alkoxy-1-
arylmethane phosphonates are used, and the labile
substituent, or its precursor, is attached to the ring at
a position so that the total number of ring atoms, e.g.,
ring carbon atoms, including the carbon atoms at the
points of attachment of the methane phosphorate group and
said labile substituent, is an odd whole number, prefer-
~ably 5 or greater, chemiluminescent 1,2-dioxetanes so
produced, when decomposed in an appropriate environment,
emit red-shifted light of greater intensity and longer
duration than when the rings are otherwise substituted.
Other substituents can be included anywhere on the
aromatic ring of these phosphonates, but at least one
substituent which can be elaborated to a chemically or
enzymatically cleavable moiety preferably is present in a
meta, or odd position relative to a "benzylic" carbon
atom which is further substituted by an alkoxy, aralkoxy,
or an aryloxy group and the phosphorous atom of the phos-
phonate ester group.
An example of an elaboratable group is the bromine
atom in diethyl 1-methoxy-1(3-bromophenyl)methanephos
phonate, which upon Horner-Emmons reaction with a T - 0
ketone yield an enol ether, e.g..
~3
Et
... . .. .. . . ... . ..._.. ~..:.~ 02340484 2001-03-19 r~ r;i;.~ .' ' . _ ,.
_.,...,... , ,
~s~:.:....._.......~._ ~,..,~,~._._~.,_.._...._._..._,...~_..,.._.
8
This enol ether can be converted to a Grignard reagent
or an organolithium derivative for reaction with
elemental sulfur, dimethyl disulfide, or methyl .
methylthiomethyl-sulfoxide to furnish the corresponding
enol ether thiophenol or its methyl ether. The same
organometallic species can be reacted with
trimethylsilyl azide or azidomethyl phenyl sulfide
[Tanaka, N., g~ ~, J.C.S. Chem. Comm., 1322 (1983):
Trost, B., g~ ~, J. Am. Chem. Soc., 103:2483 (1981)]
to give the aminophenyl enol ether or its N-acyl or
sulfonamide derivatives.
It has also been discovered that it is often times
advantageous to conduct the acylation reaction of Step 7
in the above-described reaction sequence, or the
phosphorylation reaction of Step 8, or the glycosylation
reaction of Step 11, using hydroxyaryl enol ether alkali
metal salts of the formula:
OR3
T
2 0 ( X~~--~ Y O~ AM'
wherein AM', the alkali metal cation, is lithium sodium
or potassium and T, R3, X~ and Y are as described above,
in place of the corresponding free hydroxy compounds
depicted as compounds j, the products of Steps 6a and
6b, in this reaction sequence. In certain cases the use
of an alkali metal salt of the enol ether rather than
the free hydroxy compound results in savings in
materials of reaction. For example, acylation of the
alkali metal salt of an enol ether by the method of Step
7 above, or phosphorylation of the alkali metal salt by
the method of Step 8, preferably proceeds without using
a Lewis base in either case. In other instances there
is an actual reduction in reaction steps. Simply
employing the reaction conditions described above for
CA 02340484 2001-03-19
9
Steps 6a and 6b but dispensing with post-reaction protic
work-up, for example, will give the enol ether as its
alkali. metal salt rather than as the free hydroxy
compound. Hence, the alkali metal salt need not be
obtained by first isolating the free hydroxy compound
and then forming the salt in a separate reaction.
Instead, the thus-obtained alkali metal salts can be
separated by precipitation or used ~ situ as starting
materials for the acylation, phosphorylation or
glycosylation reactions.
It is thus an object of this invention to provide a
facile, inexpensive, high-yield, convergent chemical
synthesis of stable, water-soluble, chemically,
thermally and enzymatically decomposable,
chemiluminescent 1,2-dioxetanes such as those of formula
(I) above, by a route that employs substituted
arylaldehyde alkyl and cycloalkyl acetals and novel
phosphonate derivatives capable of forming
phosphonate-stabilized carbanions as intermediates in
the formation of the enol ether precursors of such
1,2-dioxetane end products.
It is a further object of this invention to provide
methods for synthesizing the individual substituted
arylaldehyde alkyl and cycloalkyl acetals, phosphonate
derivatives and enol ether intermediates employed in
synthesizing chemiluminescent 1,2-dioxetanes in
accordance with this invention.
It is yet another object of this. invention to
provide as novel compositions of matter substituted
arylaldehyde alkyl and cycloalkyl acetals, phosphonate
derivatives and enol ether intermediates.useful in the
synthesis of chemiluminescent 1,2-dioxetanes.
It is still another object of this invention to
provide variations in the new synthesis of stable,
waster-soluble chemiluminescent 1,2-dioxetanes disclosed
and claimed in PCT WO 88/00695, published January 18, 1988.
r
CA 02340484 2003-04-23
Another object of this invention is to provide methods
for obtaining enol ether alkali metal salt intermediates
useful in the acetylation, phosphorylation and glyco-sylation
reactions disclosed and claimed in, PCT WO 89/06226, published
July 13, 1989.
A further object of this invention is to provide methods
for obtaining and using such enol ether alkali metal salt
intermediates that result in savings in materials of
reaction, reductions in reaction steps, or both.
These and other objects of this invention, as well as a
fuller understanding of the advantages thereof, can, be had
by reference to the following disclosure.
In accordance with this invention there is provided a
compound having the formula:
UR3
O R4
wherein
O
a) R3 is methyl and R4 is -C-C (CH3) 3; or
b) R3 is a substituted or unsubstituted (i) lower alkyl,
aralkyl, or heteroaralkyl group having up to 20 carbon atoms,
(ii) aryl or heteroaryl group having up to 14 carbon atoms,
(iii) alkoxy (lower) alkyl group, (iv) a hydroxy (lower) alkyl
CA 02340484 2003-04-23
10a
group having up to 6 carbon atoms, a (lower) alkyl-OSiX3 group
wherein the lower alkyl group contains up to 6 carbon atoms
and any X is independently methyl, phenyl, or t-butyl, or (v)
amino (lower) alkyl or mono or di(lower) alkylamino alkyl
group where each lower alkyl group contains up to 7 carbon
atoms; and
R4 is a substituted or unsubstituted allyl, benzyl or o-
nitrobenzyl group having up to 20 carbon atoms or a pivaloyl
group; or
c) R3 is a substituted or unsubstituted (i) lower alkyl,
aralkyl, or heteroaralkyl group having up to 20 carbon atoms,
(ii) aryl or heteroaryl group having up to 14 carbon atoms,
(iii) alkoxy (lower) alkyl group, (iv) a hydroxy (lower) alkyl
group having up to 6 carbon atoms, a (lower) alkyl-OSiX3 group
wherein the lower alkyl group contains up to 6 carbon atoms
and any X is independently methyl, phenyl, or t-butyl, or (v)
amino (lower) alkyl or mono or di(lower) alkylamino alkyl
group where each lower alkyl group contains up to 7 carbon
atoms; and
R4 is hydrogen.
Detailed Description of the Invention
The 1,2-dioxetanes, and in particular the enzymatically-
cleavable dioxetanes in which T is a spiro-bonded
substituent, a c~em_ carbon of which is also the 3-carbon atom
of the dioxetane ring, disclosed and claimed in the
aforementioned publications of Bronstein, Bronstein et al,
Edward and Edwards et al, and their thermally, chemically and
electrochemically cleavable analogs, form one class of water-
soluble chemiluminescent 1,2-dioxetane compounds that can be
synthesized by the method of this invention. These
CA 02340484 2003-04-23
lOb
1,2-dioxetanes can be represented by formula (I) above, T
being a stabilizing group. The most preferred stabilizing
group is a fused polycycloalkylidene group bonded to the
3-carbon atom of the dioxetane ring through a spiro linkage
and having two or more fused rings, each having from 3 to 12
carbon atoms, inclusive, e.g., an adamant-2-ylidene, which
may additionally contain unsaturated bonds or l,2-fused
aromatic rings, or a substituted or unsubstituted alkyl group
having from 1 to 12 carbon atoms, inclusive, such as tertiary
butyl or 2-cyanoethyl, or an aryl or substitutes aryl
. .... .... .___.._....:.::~...~.CA 02340484 2001-03-19 .,~ . :: ,
.........,.....>..<. _",~,;_.-.m.:...,. __...,... ...._._.~,... ..
11
group such as carboxyphenyl, or a halogen group such as
chloro, or heteroatom group which can be a hydroxyl
group or a substituted or unsubstituted alkoxy or
aryloxy group having from 1 to 12 carbon atoms,
inclusive, such a,s an ethoxy, hydroxyethoxy,
methoxyethoxy, carboxymethoxy, or polyethyleneoxy group.
The symbol R3 represents a C~-CZO unbranched or
branched, substituted or unsubstituted, saturated or
unsaturated alkyl group, e.g., methyl, allyl or
isobutyl; a heteroaralkyl or aralkyl (including
ethylenically unsaturated aralkyl) group, e.g., benzyl
or vinylbenzyl: a polynuclear (fused ring) or
heteropolynuchear aralkyl group which may be further
substituted, e.g., naphthyl-methyl or 2-benzothiazol-
2-yljethyl: a saturated or unsaturated cycloalkyl group,
e.g., cyclohexyl or cyclohexenyl: a N, 0, or S
heteroatom containing group, e.g, 4-hydroxybutyl,
methoxyethyl, or polyalkyleneoxyalkyl; an aryl group,
any of which may be fused to Y such that the emitting
fragment contains a lactose ring, or an enzyme-cleavable
group containing a bond cleavable by an enzyme to yield
an electron-rich moiety bonded to the dioxetane ring;
preferably, X is a methoxy group.
The symbol Y represents a light-emitting
fluorophore-forming group capable of absorbing energy to
form an excited energy state from which it emits
optically detectable energy to return to its original
energy state. Preferred are phenyl, biphenyl, ,
9,10-dihydrophenanthryl, naphthyl, anthryl, pyridyl,
3o quinolinyl, isoquinolinyl, phenanthryl, pyrenyl,
coumarinyl,~carbostyryl, acridinyl, dibenzosuberyl,
phthalyl or derivatives thereof.
The symbol Z represents hydrogen (in Which case the
dioxetane can be thermally cleaved by a rupture of the-
oxygen- oxygen bond), a chemically-cleavable group such
as a hydroxyl group, an alkanoyloxy or aroyloxy estet
group, silyloxy group, cr an enzyme-cleavable group
CA 02340484 2001-03-19
12
containing a bond cleavable by an enzyme to yield an
electron-rich moiety bonded to the dioxetane ring, e.g., a
bond which, when cleaved, yields a Y-appended oxygen anion, a
sulfur anion, an amino or substituted amino group, or a
nitrogen anion, and particularly an amido anion such as
sulfonamido anion.
One or more of the substituents T, R3 and Z can also
include a substituent which enhances the water solubility of
the 1,2-dioxetane, such as.a carboxylic acid, e.g., a carboxy
methoxy group, a sulfonic acid, e.g., an aryl sulfonic acid
group, or their salts, or a quaternary amino salt group,
e.g., trimethyl ammonium, with any appropriate counter ion.
When using an enzymatically-cleavable 1,2-dioxetane,
cleavage can be accomplished using an enzyme such as alkaline
phosphatase that will cleave a bond in, for example, a Z
substituent such as a phosphate mono ester group, to produce
a Y oxy-anion of lower oxidation potential that will, in
turn, destabilize the dioxetane and cleave its oxygen-oxygen
bond. Alternatively, catalytic antibodies may be used to
cleave the Z substituent. Destabilization can also be
accomplished by using an enzyme such as an oxido-reductase
enzyme that will cleave the oxygen-oxygen bond directly: see
the aforementioned PCT WO 88/00695, published January 28,
1988.
Besides a phosphate ester group, Z in formula I above
can be an enzyme-cleavable alkanoyloxy group, e.g., an
acetate ester group, an oxacarboxylate group, or an
oxaalkoxycarbonyl group, 1-phospho-2,3-diacylglyceride group,
1-thio-D-glucoside-group, adenosine triphosphate analog
group, adenosine diphosphate analog group, adenosine
monophosphate analog group, adenosine analog group, a-D-
galactoside group, ~-D-galactoside group, a-D-glucoside
group, ~-D-glucoside group, a-D-mannoside group, ~-D-
mannoside group, ~-D-fructofuranoside group, ~-D-
glucosiduronate
CA 02340484 2001-03-19 ..
13
group, an amide group, p-toluene sulfonyl-L-arginine
ester group, or p-toluene sulfonyl-L-arginine amide
group.
The method for producing 1,2-dioxetanes according
to this invention can be illustrated in part by the
following reaction sequences leading to the preparation
of 1,2-dioxetanes having both an alkoxy (or aryloxy) and
an aryl substituent at the 4-position in which the
latter (illustrated here as an aryl Y substituent) is
itself substituted by one or more X' groups, these
substituents being , meta, or para to each other.
As will be appreciated by one skilled in the art, groups
RZ or X' need not be static during the reaction
sequences, but may be interconverted under conditions
which are compatible with structural considerations at
each stage.
CA 02340484 2001-03-19
14
CHO (III)
X~-~'-R=
R30H
2. V1'ater scavenger
Catal~~st
PQ3 = 3R~OH
1. Base
R'O~OR'
(R~O)3P X~-7~'-R=
3. Lew~is acid
O ORS
RlO H PI/
X~-~.-R= ORS
1) base. solvent, cold
4. ') T = O.
O R'
T
X 1-7.-R.
5. ~OZ, by
O- O OR3
T
X ~-Y-R'-
CA 02340484 2001-03-19 '.. . . ,.,__ .., _
In these formulae: any Q can be independently a
halogen, e.g., chlorine or bromine, or OR': R' can be
independently a trialkylsilyl group or a lower alkyl
group having up to 12 carbon atoms such as ethyl,
5 propyl, or butyl; RZ can be a hydroxyl group, an ether
(0R4) or a thioether (SR4) group wherein R4 is a
substituted or unsubstituted alkenyl, lower alkyl or
aralkyl group having up to 20 carbon atoms such as
methyl, allyl, benzyl, or o-nitrobenzyl; RZ can also be
l0 an acyloxy group such as acetoxy, pivaloyloxy, or
mesitoyloxy, a halogen atom, e.g., chlorine or bromine,
a vitro group, an amino group, a mono or di(lower) alkyl
amino group or its acid salt wherein each lower alkyl
substituent contains up to 7 carbon atoms such as
15 methyl, ethyl, or butyl, where any or all of these lower
alkyl groups may be bonded to Y generating one or more
fused rings, a NHSOZRS group wherein RS is methyl, tolyl,
or trifluoromethyl;'RZ can also be a substituted aryl,
heteroaryl, ~-styreneyl group containing up to 20 carbon
atoms such as a 4-methoxyphenyl, or 6-methoxy-
benzthiazol-2-yl group; R3 can be a substituted or
unsubstituted lower alkyl, aralkyl, or heteroaralkyl
group having up to 20 carbon atoms such as methyl,
trifluoroethyl, or benzyl, an aryl or heteroaryl group
having up to 14 carbon atoms which may be further
substituted, e.g., a 4-chlorophenyl group, a (lower)
alkyl-OSiX3 group wherein the lower alkyl group contains
up to 6 carbon atoms such as ethyl, propyl, or hexyl and
any X is independently methyl, phenyl, or t-butyl, an
alkoxy (lower) alkyl group such as ethoxyethyl, or
ethoxypropyl, a hydroxy (lower) alkyl group having up to
6 carbon atoms such as ethyl, butyl, or hexyl, or an
amino (lower) alkyl or sono or di(lower) alkylamino
alkyl group where each lower alkyl group contains up to
7 carbon atoms such as methyl, ethyl, or benzyl; X~ can
be hydrogen or a substituted or unsubstituted aryl,
aralkyl, heteroaryl, or heteroaralkyl group having up to
...... ..._..__.CA 02340484 2001-03-19 . ..: . _
16
20 carbon atoms such as 4,5-diphenyloxazol-2-yl,
benzoxazol-2-yl, or 3,6-dimethoxy-9-hydroxyxanthen-9-yl
groups, an allyl group, a hydroxy (lower). alkyl group
having up to 6 carbon atoms such as hydroxymethyl,
hydroxyethyl, or hydroxypropyl, a (lower) alkyl-OSiX3
group wherein the alkyl and X radicals are as defined
above, an ether (ORS) or a thioether (SR4) wherein R4 is
as defined above, an SOiR6 group wherein R6 is methyl,
phenyl, or NHCbIis, a substituted or unsubstituted alkyl
group containing up to 7 carbon atoms such as methyl,
trifluoromethyl or t-butyl, a vitro group, a cyano
group, an aldehydic function or its oxime or
dimethylhydrazone, an alkyl halide group having up to 6
carbon atoms and the halide group being chlorine or
bromine, a halogen group, a hydroxyl group, a carboxyl
group or its salt, ester or hydrazide derivatives, a
tri-substituted silicon-based group such as a
trimethylsilyl group, or a phosphoryloxy (phosphate
monoester) group.
Step 1 of the foregoing reaction sequence involves
the formation of a tertiary phosphorous acid alkyl ester
from a phosphorous trihalide, e.g., phosphorous
trichloride or dialkylchlorophosphite; and an alcohol,
e.g., a short chain alkyl alcohol, preferably one having
up to 7 carbon atoms such as methanol, ethanol or
butanol, in the presence of a base such as
triethylamine. An alkali metal alcoholate or
trialkylsilanolate can also be used in a direct reaction
with the chlorophosphite.
Step 2 involves reacting an aryl aldehyde or
heteroarylal.dehyde with an alcohol, R30Fi, to give the
corresponding aryl aldehyde acetal, wherein the aryl
aldehyde may be a benzaldehyde, a naphthaldehyde, a
anthraldehyde and the like, or aryl dialdehydes such as
m-or p-phthalaldehydes and the like. The RZ substituent
on the aryl aldehyde, which is preferably positioned~
meta to the point of attachment of the aldehydic group
. ~ 02340484 2003-04-23 --.
..... ~ . . . ..w.. .. ..... .. ~.___..._...~.__....~..._..,...,.......-
.~rbw.~;tv.~.bet..;~,.n.<..i:x~.ray..b6rn...fev:ae_.xa..~a~.m ~.... . . v. ...
in the benzaldehydes illustrated above, can be an
oxygen-linked functional group, e.g., an ester group
such as pivaloyloxy, acetoxy and the like, an ether
group such as methopy, benzyloxy, and the like, a vitro
group, a halogen atom, or hydrogen (see Tables 2-6
below). Functional group X' in the aryl aldehyde may be.
located ortho, metes or to the point of attachment
of the aldehydic group to the aryl ring, and can be a .
lower alkoxy group such as methoxy, ethoxy or the like,
hydrogen, or an alkyl group (see Table 2 below). In the
alcohol reactant R3oH, R3 can be, for example, a lower
alkyl group such~as methyl, ethyl and the like, a lower
aralkyl group, a lower alkoxy alkyl group-, a substituted
amino alkyl group, or a substituted siloxy alkyl group
(see Tables 2-6j. Diols such as ethylene glycol or
propylene glycol, e.g., Ii0-(CHZ)~ OH, produce cyclic
acetals which are within the scope of this invention.
The acetalization reaction between the aryl aldehyde and
the alcohol or diol is carried out in conventional
fashion, preferably in the presence of a catalyst such
as a Lewis acid, HCl(g), p-toluenesulfonic acid or its .
polyvinylpyridine salt, or A~aberlyst1010 resin,
accompanied by removal of water using, e.g.,
trialkylorthoformate, 2,2-dialkoxypropane, anhydrous
copper sulfate, or molecular sieves, or by azeotropic
distillation in, for example, a Dean-Stark apparatus.
In cases in which acetalization may proceed with poor
conversion or yield, it is possible to use the Noyori
reaction wherein any of the aforementioned alcohols
(R30H) or diols are reacted with the aldehyde as their .
mono or trialkylsilyl ether with trimethylsilyl
triflate as catalyst in a chlorinated hydrocarbon
solvent.
Step 3 involves reacting the tertiary phosphorous.
acid alkyl ester (trialkylphosphite) produced in Step 1
with the aryl aldehyde dialkyl or cyclic acetal produced
in Step 2, preferably in the presence of at Ieast one
CA 02340484 2001-03-19
18
equivalent of a Lewis acid catalyst such as BF3 etherate
or the like to give the corresponding phosphonate,
essentially according to Burkhouse, D.,~g~ ~, .
Synthesis, 330 (1984). Aryl aldehyde dialkyl acetals
react with between 1 and 1.5 equivalents of a
trialkylphosphite in the presence of a Lewis acid in an
organic solvent such as methylene chloride, under an
inert atmosphere, e.g., argon, at temperatures below
0'C, to produce in almost quantitative yields (see Table
2) the corresponding 1-alkoxy-1-arylmethane phosphonate
esters. The phosphonates are generally oils that can be
used directly or purified by chromatography on silica
gel. ~HNI~t spectra will exhibit a doublet near 4.7 ppm
(J = 15.5 Hz) due to the benzylic proton, split by the
adjacent phosphorous atom; occasionally, two doublets
of unequal intensity will be observed.
In step 4, the phosphonate-stabilized carbanion is
used to synthesize olefins by the Horner-Emmons
reaction. Specifically, in Step 4.1 a phosphonate-
stabilized carbanion is produced from a dialkyl
l~alkoxy-1-arylmethane phosphonate in the presence of a
base such as sodium hydride, sodium amide, a lithium
dialkyl amide such as lithium diisopropylamide (LDA), a
metal alkoxide, or, preferably, n-butyllithium, in a
suitable solvent, preferably in the presence of a slight
excess of base, e.g., about 1.05 equivalents for each
. ionizable group present. Suitable solvents for the
reaction can have an appreciable range of polarities,
and include, for.example, aliphatic hydrocarbons such as
hexanes, aromatic hydrocarbons such as benzene, toluene
and xylene, ethers such as tetrahydrofuran (THF) or
glymes, alkanols such as ethanol and propanol,
dimethylformamide (DMF), dimethyl-acetamide, and
dimethylsulfoxide, and the like, or mixtures of these _
solvents. As lithiophosphonates are insoluble in
diethylether, but soluble in ethers such as THF, ~.
reactions using LDA or n-butyllithium are preferably run
CA 02340484 2001-03-19
19
in dry THF/hexane mixtures. It is also preferred to carry out
the reaction in an inert atmosphere, e. g., under argon gas.
At temperatures below 0°C the reaction of n-butyllithium with
phosphonates proceeds rapidly, as indicated by the
instantaneous formation of a dark yellow to burgundy colored
solution, depending upon the particular phosphonate used and
its concentration.
In Step 4.2, the phosphonate-stabilized carbanion is
reacted, preferably in molar excess, with a carbonyl compound
T = O or dicarbonyl compound O = T = O. When T = 0 is a
substituted or unsubstituted adamantanone,. e.g., adamantanone
itself, the reaction begins immediately upon addition of the
ketone, preferably from about 0.8 to about 0.95 equivalents
of the ketone, to the stabilized carbanion, and goes to
completion under reflux conditions in from about 2 to about
24 hours. Optimization of the T = O equivalency in each case
allows complete conversion of this expensive component.
In Step 5 the enol ether is oxidized. Oxidation is
preferably accomplished photochemically by treating the enol
ether with singlet oxygen (10z) wherein oxygen adds across the
double bond to create the 1,2-dioxetane ring. Photochemical
oxidation is preferably carried out in a halogenated solvent
such as methylene chloride or the like. 102 can be generated
using a photosensitizer, such as polymer bound Rose Bengal
(Hydron Labs, New Brunswick, N.J.) and methylene blue or 5,
10, 15, 20-tetraphenyl- 21H,23H-porphine (TPP). Chemical
methods of dioxetane formation using triethylsilyl-
hydrotrioxide, phosphate ozonides, or triarylamine radical,
cation mediated one electron oxidation in the presence of 302
can also be utilized.
When the oxygen-linked functional group R2 on the aryl
ring of the enol ether is an alkoxy group or pivaloyloxy
group, it can be converted to an enzymecleavable group such
as a phosphate, acetoxy, or O-hexopyranoside group, by
carrying out the following
CA 02340484 2001-03-19...-..._,-
.,...~....<.......__...~.~.._.......,...._..y.................._...,_.._ ,
additional steps involving the enol ether produced in
Step 4 of the foregoing reaction sequence prior to
carrying out the oxidation reaction of Step 5, as shown
below:
CA 02340484 2001-03-19
21
OR3 6b. KZC03/MeOH (IV)
T~
(XI, \Y_ ~ OR3
OR T
h 6a. NaSE: '~
aprotic solvent (XI)._y_O
H
J
O O
II / 7. A
- Q_p Lewis base
O
OR3
Lewis base
aproptic solvent
~ I)-Y-OAc
k
terra-0.
II. aceiyl- OR3
D-hezo. T-~ O p
PYranosyl II /
halide X I'-Y-'O-P
1 O
OR3
T
MCw
XI-~'-O'-D-hexopYrano- 9. solvent
side tetraacetate
0
O R'
T~ ~O-M-
I.. Base X I_ y-O_ p
OR' m O(CH=)ACV
T~ 1p. BOCH: or an
X~y_O_ ammonium base. BOH
D-
hexopyranoside '
P
OR z
Z.~ ~ O _ y,I
X L_ Y._ O_ P
O-B+
n
CA 02340484 2001-03-19
22
Step 6a. involves aryl ether cleavage of the R~
substituent (wherein R~ is preferably methyl, allyl or
benzyl), preferably with sodium thioethoxide, in an
aprotic solvent such as DMF, NMP, or the like, at
temperatures from about 120'C to about 150'C. The
cleavage can also be accomplished with soft nucleophiles
such as lithium iodide in refluxing pyridine, sodium
cyanide in refluxing DMSO, or Na2S in refluxing
N-methyl-2-pyrrolidone. When R7 is pivaloyl, ester
l0 cleavage can be accomplished with NaOMe, KOH or KZC03 in
an alcoholic solvent such as MeOH at temperatures from
about 25'C to reflux (Step 6b.).
The acylation of the aryl hydroxyl group in the
thus~obtained hydroxy compound is carried out in Step 7
by adding a small equivalent excess of an acid halide or
anhydride, e.g., acetic anhydride, or oxalyl chloride
with Lewis base, e.g., triethylamine, in an aprotic
solvent. '
The substituent Q on the cyclic phosphorohalidate
used in Step 8 is an electronegative leaving group such
as a halogen. The monovalent cation M' of the cyanide
used in Step 9 can be a metallic or alkali metal cation
such as Na' or K', or a quaternary ammonium cation. The
cation B' of the ammonium base of Step 10 is an ammonium
cation: however, NaOMe can also be used as the base. T,
R3 and X' are as def ined above.
Steps 8, 9 and 10 can be performed separately or in
a onepot or two-pot operation. A cyclic phosphoroha-
lidate, e.g., cyclic phosphorochloridate, is preferred
for use in Step 8 not only because of its monofunction-
ality, chemoselectivity and enol ether-compatible
deprotection mode of action, but also because it is 106
times more reactive than the corresponding acyclic
compounds. In a 3-step, 2-pot operation, the aryl;:
hydroxyl group in the free hydroxyl product produced in
Step 6 is zeacted with 2-halo-2-oxo-1,3,2-dioxaphospho-
lane to yield the cyclic phosphate triester (Step 8).
CA 02340484 2001-03-19
23
This triester is subjected to ring opening with MCN (e. g.,
NaCN) to.yield the corresponding 2-cyanoethyl diester (Step
9). A base, e.g., ammonium hydroxide or~NaOMe, then provokes
a facile (3-elimination reaction, yielding a filterable
disodium sodium ammonium salt (Step 10). In benzene, THF,
diethylether or DMF, phosphate triester formation induced by
a Lewis base (e. g., a tertiary amine such as triethylamine)
or with a preformed alkali metal salt of the phenolic
enolether cane effected with phosphorohalidates over a
temperature range of about -30° to about 60°C. Subsequently,
if a pure monosodium cyanoethylphosphate ester is desired,
the ring cleavage with alkalicyanide (MCN) in DMF or DMSO can
be carried out in a narrow temperature range of between about
15° and about 30°C. However, in a one-pot or in situ mode
this is not as important, and the temperature range widens to
about 60°C on the high end.
Aryl phosphate disalts can also be made from the aryl
alcohol enol ether product of Step 6 (formula IV) using an
activated phosphate triester of the general formula:
O/O~ R8 O/OSi(CH3)3
Q-P or Q-P
\O~ RQ \OSi(CH3)3
wherein Q is as described above, and Re and R9 are each
independently-CN, -NOz, arylsulfonyl, or alkylsulfonyl.
Alternatively, the phosphate triester may contain two
trimethyl silyl groups, linked to the phosphorous, as shown
in the formula above. This reaction can be carried out in the
presence of a Lewis base in an aprotic solvent, and yields an
aryl phosphate triester. The triester can then be hydrolyzed
with a base, M"OH or M' OCH3, wherein the cation M' is an
alkali metal, NR1~4'
CA 02340484 2001-03-19
24
wherein R'° is hydrogen or a C~-C~ alkyl, aralkyl, aryl or
heterocyclic group, to give the corresponding
arylphosphate monoester disalt via p-elimination.
Dioxetane formation of the reaction of singlet oxygen
('OZ) with these enol ether phosphate triesters, followed
by similar base-induced deprotection to the dioxetane
phosphate monester, may also be carried out.
An alkoxy group on the aryl ring of the enol ether
can be converted,to a D-sugar molecule linked to the
ring via an enzyme cleavable glycosidic linkage by
reacting the phenolic precursor in an aprotic organic
solvent under an inert atmosphere with a base .
such as ivaH~,: and .. with a tetra-O-acetyl-D-hexopyranosyl
halide to produce the aryl-O-hexopyranoside tetraacetate
(Step 11). The protective acetyl groups can then be
hydrolyzed off using a base such as NaOCH3, KZCO3, or NH3
gas, in an alcohol such as methanol, first at 0'C and
then at 25'C for 1 to 10 hours (Step 12), leaving a
hexosidase-cleavable Dhexopyranosidyl moiety on the aryl
ring.
When the enol ether aryl phosphates are oxidized to
the _ __ "corresponding 1,2-dioxetanes
(Step 5 above), ion exchange to a bis-guaternary
ammoniun or monopyridinium salt allows the facile
photooxygenation of 0.06 M chloroform solutions in the
presence of, preferably, methylene blue or TPP, at cold
temperatures, e.g., about 5'C. Slower reaction rates
and increased photolytic damage to the product may occur
with the use of solid phase sensitizers such as
polymerbound Rose Bengal (Sensitox I) or methylene blue
on silica gel.
Aryl monoaldehydes or heteroaryl monoaldehydes
other than those having formulas such as:
CHO
~1
CA 02340484 2001-03-19
can also be used as starting materials in carrying out
the above described reaction sequences. Included among
such aryl monoaldehydes are polycyclic aryl or
heteroaryl monoaldehydes such as those having the
5 formula
CHO
R~
wherein RZ is as defined above and is preferably
positioned so that the total number of ring carbon atoms
separating the ring carbon atom to which it is attached
and the ring carbon atom to which the aldehyde group is
10 attached, including the ring carbon atoms at the points
of attachment, is an odd whole number, preferably 5 or
greater; see ~dwards, g~ ~, U.S. Patent 4,952,707,
issued August 2$, 1990.
Fused heterocyclic acetals or hemiacetals can also
15 be used as starting materials in carrying out the
above-described reaction sequences. Included among such
fused heterocyclic acetals are those having the
formulae:
W 2
R2 ~ (R )
O
. tC~2)n
0 0 ~~'~ "1'0~0'~
R2
CA 02340484 2001-03-19
26
and the like, wherein R~ is as described above, and, W can be
OR', wherein R' is described above, or OH, and n is an integer
greater than zero.
Aryl or heteroaryl dialdehydes can also be used as the
aldehydic starting material, e.g., ones having the formula:
OHC CHO
_ .
Rz
wherein Rz is as described above.
Purification of the thus-obtained water-soluble
dioxetanes is best achieved at alkaline pH values, e.g.,
about 7.5 to about 9.0, using reverse phase HPLC with an
acetonitrile-water gradient, followed by lyophilization of
the product.
Typical enzymatically-cleavable water-soluble
chemiluminescent 1,2-dioxetanes for use in bioassays which
can be prepared by the method of this invention are the 3(2'-
spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy) phenyl-1,2-
dioxetane salts represented by the formula:
O - O OCH (~.)
3
OPO; =(:r! y )z
o
CA 02340484 2001-03-19
27
wherein M' represents a cation such as an alkali metal, e.g.
sodium or potassium, or a C1-C18 alkyl, aralkyl or aromatic
quaternary ammonium cation, N (R1°) 4+, in which each R1° can be
alkyl, e.g., methyl or ethyl, aralkyl, e.g., benzyl, or form
part~of a heterocyclic ring system, e.g., N-methylpyridinium,
a fluorescent onium cation, and particularly the disodium
salt. A more systematic name for the latter is 3-(4-
methoxyspiro[1,2-dioxetane-3,2'-tricyclo[3.3.1.13'']decan]4-
yl)phenylphosphate disodium salt. -
The availability of the herein described Horner-Emmons
methodology and a pool of reactants containing the particular
aforementioned class of mono and bis-phosphonate esters along
with T = 0 ketones or O = T = O diones such as 2,6-
adamantanedione, allows the synthesis of three different enol
ether product types. (formula VI)
OR3 (VI)
T
Y-Z
R-~O O R 3
~T~ '
Z-Y/ Y-Z
0
R30 OR3
T~ ~=T
_ ~,
Z
C
wherein T, R', Y and Z are as described herein above. These
can then be converted to the corresponding 1,2-dioxetanes
shown below in formula (VII) with singlet oxygen as described
herein above.
CA 02340484 2001-03-19
2s
O-O ORS (~.'II)
T
R'O OR3
0 0_0 ; o_ o
Z
°_o °_o
T
RiO~\ / ~ORz
- _ Z_Y Y-Z _
In the case of 1,2-dioxetane B of formula (VII), one T
group serves to stabilize two dioxetane rings: however, each
ring must be destabilized individually by chemical or
enzymatic means at each Z group. In 1,2-dioxetane C, one Z
group can activate the decomposition of two dioxetane rings,
especially if all groups appended to aromatic ring Y are
disposed in a meta or odd-pattern relationship with one
another as described above.
The bis-enol ether phenol of formula (VIII) below~is
synthesized by sodium ethane thiolate cleavage of the
aromatic methoxy group (Step 5 of the flow chart (III)) of
the compound described in Examples 62 and 105 below. The
product can be converted to any one of the enzyme cleavable
groups described above, e.g., a phosphate mono ester. As such
it represents a pivotal intermediate for the synthesis of
1,2-dioxetanes of type C of formula (VII) as shown above.
(Z'III)
O-O
~ ORi R30 O _ O ocH; ocH;
T ~ O I
_ Il ~- T I \
Y O
I~O-Y \
OH
A modified method of providing the enol ether alkali
metal salts of this invention involves
~ 02340484 2001-03-19 ",_._...,.,..,__....._.,_. ...__._.__._ .._ . _...._. .
.. . .
29
modification of the step in the above-described reaction
followed by modification of the subsequent ester
cleavage step, Step 6b. Specifically, and as described
above, in the first part of this modified procedure a
dialkyl 1-alkoxy-1-arylmethane phosphonate:
jl/cr~
a w.
~_x- ~
preferably one in which Y is an aryl moiety, e.g, a
phenyl ring, RZ is an acyloxy substituent, preferably in
the meta-position on the aryl moiety, e.g., a
pivaloyloxy group, and X' can be hydrogen or another of
the substituents listed above, is converted to the
corresponding phosphonate-stabilized a-carbanion,
preferably in solution at low temperature, -20'C or
less, under an inert atmosphere, using an alkali metal-
containing base, e.g., from about 1 to about 1.2
equivalents of the alkali metal-containing base, and
preferably slightly more than one equivalent of an
alkali metal alkylamide such as lithium diisopropyl-
amide or an alkali metal alkyl compound such as
n-butyllithium.
Once the a-carbanion is formed the polycyclic
ketone T = O is added to the reaction mixture at low
temperature, preferably in slightly less than molar
excess, and reacted under reflux conditions for from
about 2 to about 24 hours to give a reaction mixture
which can include, inter alia, the dialkyl 1-alkoxy-1-
arylmethane phosphonate starting material as its anion,
its R2 deesterified dianion, or its decomposition
products, the hydroxyaryl enol ether alkali metal. salt,
and the RZ esterified aryl enol ether, the latter
particularly beiaq present irher~ the. phosphonate stazting
material inclines an r~rylcxy-substituted aryl moiety..
(Y - R~j whese acyloxy substituent (Rtj has nn acyi group
CA 02340484 2001-03-19
that is a good hydroxy protecting group that remains
substantially intact during this reaction, e.g., a pivaloyl
group (R2 - pivaloxyloxy). It has been found, in fact, that
when the phosphonate starting material's Y - Rz substituents
5 constitute a pivaloyloxyphenyl group, only about 10-20
percent of the total enol ether product obtained is present
as the deesterified enol ether alkali metal salt.
Mild protic work-up of this reaction mixture to separate
the desired ~ esterified aryl enol ether is complicated by
10 the presence of several other useful components, all which
should, if possible, be recovered in some fashion to reduce
costs. The RZ esterified aryl enol ether where Rz is a
pivaloyloxy group, for example, is a high Rf, early eluting
product when subjected to column chromatography, while the
15 corresponding hydroxyaryl (deesterified) compound, which is
produced during protic work-up to from the hydroxyaryl enol
ether lithium salt, and the phosphonate starting material and
its decomposition products, are somewhat lower Rf materials,
making for a difficultly separable mixture which yields
20 somewhat impure fractions on a large synthetic scale.
Reesterification of the crude, post-reflux Horner-Emmons
reaction mixture, however, to substantially esterify the
hydroxyaryl enol ether alkali metal salt, preferably using an
acid chloride or acid anhydride, e.g., pivaloyl chloride, in
25 at least a molar equivalent amount to the total amount of all
aryloxide alkali metal salt present, permits facile
separation of_the esterified aryl enol ether in near
quantitative yield without the above-mentioned complications
during chromatography because the hydroxyaryl enol ether is
30 absent after protic workup.
The minimum quantity of acid halide or anhydride to
consume the hydroxyaryl alkali metal salt is added in
CA 02340484 2001-03-19
31
several aliquots to the crude reaction mixture, at a
temperature between about 0°C and about 50°C, over a period
of from about 2 to about 24 hours, using thin layer
chromatography to monitor the completeness of the reaction.
Where Rz is a pivaloyloxy group one gets a much cleaner
product, isolated from the reesterified mixture as a
crystalline solid using standard techniques, such.as
recrystallization from hexanes. The mother liquors,
uncontaminated with free hydroxyaryl enol ether, are easily
plug chromatographed on a large scale, again due to the
absence of hydroxyaryl enol ether byproduct.
The final reaction in this preferred method of providing
enol ether alkali metal salts involves carrying out ester
cleavage to give, instead of the free hydroxy aryl enol ether
obtained as in Step 6b of the reaction sequence set out
supra, the corresponding alkali metal salt. The salt-forming
reaction is preferably carried out using about one molar
equivalent of an alkali metal alkoxide, e.g., sodium
methoxide, in a lower alkanol, e.g., methanol or enthanol,
under anhydrous conditions, i.e., in the presence of as low
an amount of moisture as can practicably be achieved, for
from about 1 to about 4 hours at room temperature (about
25°C), followed by removal of the volatiles from the reaction
mixture in vacuo (1 mm Hg) with heating at from about 35°C to
about 65°C for about 24 hours to give the hydroxyaryl enol
ether alkali metal salt as a dry solid, directly usable in an
acylation, phosphorylation or glycosylation reaction. For
example, the free hydroxy enol ether starting material as
discussed in PCT WO 89/06226; published July 13; 1989 -- 3-
(methoxy-tricyclo[3.3.1.13'']dec-2-ylidene-methyl)phenol --
can be replaced with its sodium salt -- sodium 3-(methoxytri-
cyclo[3.3.1.1''']dec-2-ylidenemethyl)phenoxide -- in a one pot
reaction with between about 1 and 1.2 equivalents of 2-
chloro-2-oxo-1,3,2-dioxaphospho-lane in
......... .. .. .. ._:~ 02340484 2001-03-19
:.:.,.."".....:..,..~.,:,,...:_.:._r,..:~.,...-._ . ._....,.... .. . . ...
32
anhydrous dimethylform-amide or dimethylsulfoxide to'
give the corresponding cyclic triester. This triester
readily undergoes ring opening with sodium methoxide,
and ~-elimination with sodium hydroxide or ammonium
hydroxide to give the phosphate monoester salt.-
Alternatively, the same reaction can be carried out
in a halogenated solvent, e.g., methylene chloride, a
polar solvent, e.g., acetonitrile, or an ether or
polyether solvent, e.g., tetrahydrofuran or diglyme, in
the presence, if desired, of hexamethylphosphoramide or
a phase transfer catalyst such as tetrabutylammonium
bisulfate, with the remaining ring opening and
~-elimination steps being~run in dimethylformamide or
dimethylsulfoxide. These same procedures can also be
used when reacting the enol ether alkali metal salt with
the other phosphorylating agents listed above, except
that the.-elimination or hydrolysis reactions can be
run immediately following triester formation.
The enol ether alkali metal salts of this invention
can be obtained by yet another modification in the
above-described reaction sequence, this time to Step 4
alone. A dialkyl 1-alkoxy-1-arylmethane phosphonate,
Formula ~ above, whose aryl moiety (Y) has an acyloxy
substituent (RZ) the acyl group of which is a poor
hydroxy protecting group, i.e., one that will be
substantially cleaved during this reaction, such as an
acetyl group or the like, can be reacted with three
equivalents of a lithium alkyl compound, e.g.,
n-butyllithium, in solution under an inert atmosphere at
low temperature, -20'C or less, to give the correspond-
ing phosphonate-stabilized a-carbanion as its lithio
salt. Addition of the polycyclic ketone T = O,
preferably in less than a molar equivalent quantity, to
the reaction mixture, followed by refluxing for from -
about 2 to about ~1 haura,~gives the lithio salt of the
hydroxyaryl enol ether directly.
CA 02340484 2001-03-19
33
Similarly, phenolic ether or thioether cleavage of the R'
substituent exactly as described for Step 6a in the above-
described reaction sequence, using an alkali metal-containing
reagent, initially yields the corresponding hydroxyaryl or
mercaptoraryl alkali metal salt. Instead of subjecting the thus-
obtained reaction mixture to protic work-up, the thus obtained
salt can be separated by precipitation at 0°C, preferably in the
presence of a nonsolvent such as an ether, e.g., diethyl ether,
or used in situ to accomplish direct acylation, phosphorylation
or glycosylation in the manner described in Steps 7, 8 and 11 of
the above-described reaction sequence.
The conditions under which the hydroxyaryl enol ether
alkali metal salts of this invention can be subjected to
acylation, phosphorylation or glycosylation include use of any of
the solvents mentioned above, e.g., dimethylform-amide or
tetrahydrofuran, or mixtures of these solvents, for the reaction
with the acylating, phosphorylating or glycosylating reagent over
a temperature range of about 0°C to about 60°C, preferably in
the
absence of a Lewis base.
Such chemiluminescent water-soluble dioxetanes and their
derivatives can be used in a variety of detection techniques,
such as ligand binding assays and enzyme assays. Immunoassays and
nucleic acid probe assays are examples of ligand binding
techniques, in which a member of a specific binding pair is, for
example, an antigenantibody pair, or a nucleic acid target paired
with a probe complementary to and capable of binding to all and
or a portion of the nucleic acid. The ligand:an antibody and a
nucleic acid probe, can be labeled with an enzyme and a
chemiluminescent water-soluble dioxetane used as a substrate, or
a chemiluminescent:
CA 02340484 2001-03-19 _.__.:._.._..._.:_... .. . . .
34
dioxetane can be used as a label directly and conjugated
to a ligand and activated to emit light with heat,
suitable chemical agents, and enzymes. Such assays
include immunoassays to detect hormones, such as ~-human
chorionic gonadotropin (~B HCG), thyroid stimulating
hormone (TSH), follicle stimulating hormone (FSH),
_ luteinizing hormone (LH) or the like, cancer markers,
such as alpha fetal protein (AFP), carcinoembryonic
antigen, cancer antigen CA 19-9 for pancreatic cancer,
cancer antigen CA125 for ovarian cancer, haptens, such
as digoxin, thyroxines prostaglandins, and enzymes such
as phosphatases, esterases, kinases, galactosidases, or
the like, and cell su=face receptors. These assays can
be performed in an array of formats, such as solution,
bath as a two-antibody (sandwich) assay or as a
competitive assay, in solid support such as membranes
(including Western blots), and on surfaces of latex
beads, magnetic beads, derivatized polystyrene tubes,
microtiter wells, and the like. Nucleic acid assays can
be used to detect viruses e.g. Herpes Simplex Viruses,
HIV or HTLV I and III, cytomegalovirus (CNV), human
papilloma virus (HPV), hepatitis C core virus antigen
(HB~V), Hepatitis B surface antigen (HB~V), Rotavirus, or
bacteria, e.g., campylobacter jejuni/coli, E. coli, ETEC
heat labile and stable, plasmodium falciparum, or
oncogenes, or in forensic applications using human
finger-printing probes, mono and mufti loci. The
nucleic acid detections can be performed for both DNA
and~RNA in a variety of formats, e.g., solution,
derivatizea tubes or microtiter plates, membranes (dot,
slot, Southern and Northern blots) and directly in
tissues and cells y~ in-situ hybridization. DNA and
RNA can also be detected in sequencing techniques and
histocompatibility assays using chemiluminescent -
dioxetanes. Such chemiluminescent water-soluble
dioxetanes can also be used in biosensors where the
ligand-binding reaction occurs on a surface of a
CA 02340484 2001-03-19
semiconductor layer which detects chemiluminescence as
photocurrent.
Furthermore, these dioxetanes can be used in ~n
vivo applications both for diagnostics, such as imaging
5 tumor sites when coupled to a tumor site-specific
monoclonals and other ligands, or as ~ therapeutic, such
as in photodynamic therapy to photosensitive
hematoporphyrins to generate singlet oxygen - the
cytotoxic agent. In addition, enol ethers - the
10 precursors to 1;2-dioxetanes can be used as singlet
oxygen scavengers both ~ yivo and y~ vitro, to monitor
and/or inactivate this very reactive species.
.- In order that those skilled in the art can more
fully understand this invention, the following examples
15 are set forth. These examples are given solely for
purposes of illustration, and should not be considered
as expressing limitations unless so set forth in the
appended claims.
Example 1
20 ~ethoxvisoohthaldehyde
3,5-Bishydraxymethylanisole was synthesized
according to the procedure of V. Boekelheide and R.W. '
Griffin, Jr., J. Orgy. Chem., fig,, 1960 (1969). This diol
(366 mg., 2.17 mmol) was added as a solid to a stirred
25 slurry of 3 g. crushed 3A molecular sieves and 2.5 g.
w pyridinium dichromate (6.55 mmol) in 20 ml dichloro-
methane. After 3 hours at room temperature, the mixture
.... was diluted with 40 ml ether and filtered through
celite, and washed with 2:1 ether-dichloromethane. The
30 orange filtrate Was concentrated to a solid which was
boiled with 3 x 30 ml hexanes, decanting the supernate
each time from a gummy residue. As the combined hexane
fractions cooled to room temperature, fine white needles
developed in the colorless anther liquor. Filtration
35 and drying provided 150 ag (42~) of the dialdehyde which
exhibited a melting point of 110-112'C. NNR and IR data
CA 02340484 2001-03-19
36
are listed in Tables 3 and 7. TLC showed one spot (KSF,
10% ethyl acetate: dichloromethane: Rf = 0.75). These
data support the structure:
oHc
0CH3
Rxa~le 2
4-~_ thoxy-3-methoxvbenzaldehyde
Vanillin ~(10 g., 66 mmol) in acetonitrile (100 ml)
was treated with finely-powdered, anhydrous potassium
carbonate (12 g., 87 mmol) with vigorous stirring to
yield a mobile suspension. Diethyl sulfate (1l ml, 84
mmol) was~added at room temperature. The suspension was
brought to reflux, becoming quite thick after 10
minutes, but thinning again after 20 minutes. Refluxing
was continued for 48 hours, at which point water (5 ml)
was added. After an additional 2 hours of reflux, the
mixture was cooled and treated with 500 ml ice water.
Stirring at 0' for several hours produced a granular
precipitate which was filtered off and washed with
water. Air drying afforded 11.5 g. of the product (97%).
as an off-white solid melting at 61-62.5'C. NI~t and IR
data are listed in Tables 3 and 7.
CHO
GCHQ
OEt
~ 02340484 2003-04-23 , ... . .. .. .... . .. ._... ..,...... __. _ , .. ,
.... ..
37
Rxan~ple 3
3-Methoxv-2-methylbenZaldehy~g
This compound was synthesized according to the
method of Kende, A.S., g~ ~, ~T. ~ n. t,~.~~e~i~,Soc.,
~t:1860 (1979). As seen in Tables 3 and 7, HMFt and IR
data are identical to those reported. TLC showed the
title compound to be homogeneous (KSF, 20~ CIi=CLa:
hexanes: Rf = 0.17j. The major by-product in this
reaction was 2-methoxybenzylphenysulfide (Rf = .38 under
the same conditionsj.
CMO
CN3
0CH3
example 4
m-Methoxvbenzaldehyag dimethvl aggtal
m-Anisaldehyde (204.3 g, 1.5 molj was placed in a 1
litre flask under an argon atmosphere. Trimethyl
orthoformate (191 g, 1.8 molj was added quickly,
followed by 150 ml anhydrous methanol. Amberlyst~M
XN-1010 resin (2.1 g, Aldrich Chemical Co.j, which had
been previously boiled with methanol was added. The '.
mi~cture was stirred at room temperature for 22 hours '
with the exclusion of moisture. Sodium bicarbonate (1.5
g) Was added with stirring. After 20 minutes the
mixture was filtered under vacuum into a 2 litre flask
which was placed on the rotory evaporator with the water
bath temperature at 40'C. Over 30 minutes the bath was
heated to 80' to produce a clear, colorless oil. With
magnetic stirring, the oil was pumped at 65' under
vacuum (2mm 3;gj for 30 minutes. The resulting product
weighted 272.5 g (99.8~j. I.R. (near, cm ~js 2935, ~w
2824, 1598, 1584, 1350, 1258, lLQEi~ 1050, 984, 771.
CA 02340484 2003-04-23
38
1HNMR (400 MHz, CDC13) : $ 3.33 (6H, s, OCH3) : 3.81 (3H, s,
ArOCI~) ; 5 . 35 (1H, s, ArCH (OAF ) s; 6. 87_ (1H, br d, 8 .1 Hz) t
7.00 - 7.03 (2H, m); 7.27 (1H, t, 8.lHz). These data
indicated that the product was pure enough for us.e in the
next step and were consistent With the following structure:
CHiO OCH3
~ - ~. _
OCH3
Example 5
D' th 1 1-metho -1- -m ho n a ne s hona a
m-Methoxybenzaldehyde dimethyl acetal from Example 4
(271.4 g, 1.49 mol), triethyl phosphate (250.3 g, 1.51 mol),
and methylene chloride (600 ml) were charged into a 3 litre
3-necked flask which was outfitted with a dropping funnel, an
argon inlet, and an argon outlet. The flask was flushed with
argon and the funnel was capped with a septum. The mixture
was stirred and cooled to -40° in a liquid nitrogen-acetone
bath. Soron trifluoride etherate (198.1 ml, 1.61 mol) was
then added dropwise from the funnel over a 25 minute period.
The mixture was allowed to slowly warm up to 5° over 3 hours.
Stirring was then continued at room temperature for another .
15 hours. The light yellow solution was then stirred rapidly
as 500 ml saturated sodium bicarbonate solution was added:
After 1 hour the mixture was transferred to a separatory
funnel. The organic layer was isolated and washed with 500 ml
water, 2 x 300 ml saturated sodium bisulfate, and 300 ml
.. . . ~ - . ...~ 02340484 2001-03-19 -..~.~.~__..._,..... . .. ... . .. . ,
_.~i~_.. . . . . . ~_-__._._..
39
saturated bicarbonate solution. Drying was accomplished
over 30 g anhydrous sodium sulfate just before
decolorizing carbon (3g) was added to the solution, and
the whole was filtered under vacuum through celite.
Concentration on the rotory evaporator and high~vacuum
pumping to a final pressure of 0.15 mm Hg at 100'C
provided a light yellow oil weighing 380 g (90~). I.R.
(neat, c~~): 2974, 1596, 1582, 1480, 1255 (P ~ O),
1098, 1050, 1020, 965, ~HNl~t (400 M~iz, CDC13) : d 1.21
and 1.25 (6H, two- t, 7Hz, OCHZCH3) : 3.37 (3H, s,
ArCHOC~I3); 3.80 (3H, s, ArOCg3); 3.90 - 4.10 (4H, m,
OC~-I2CH3); 4.46 (1H, d, 15.6Hz, ArCgPO); 6.85 (1H, m);
7.00 (2H, m), 7.26 (1H, m). This product was
sufficiently pure for use in a Horner-Emmons reaction.
However, further purification to remove a trace of a
non-polar fluorescent impurity may be accomplished with
silica gel chromatography using dichloromethane to elute
the impurity and subsequent elution with 20~ ethyl
acetate in dichloromethane to elute the phosphonate.
CH Q p , OF t
"~ DIt
ocx~
Example 6
~-2-Adamantylidene-a-methoxv-m-methoxvtoluene
One hundred grams of the phosphonate ester from
Example 5 (0.347 mol) were dissolved in 650 ml HPLC
grade THF (no special precautions to dry the solvent -
were taken). The solution was placed in a dry 2 litre,
3-necked flask which was outfitted with an addition
.......... .._ ...._ ... . ...._,... _....__~ 02340484 2001-03-19 ~~--
....,,...Y._._L..._.~___._. ..__...,.... ....._. ...___..__. . ...,._._._
funnel connected to an argon outlet, an argon inlet, and
a septum.
After purging with argon, the flask was lowered
into a dry ice/acetone bath at -78' and magnetic
5 stirring was initiated. After stirring for 10 minutes, a
solution of n-butyllithium in hexane (217 ml of a 1.6 M
solution, 0.347 mol) was added by syringe in several
portions over 10 minutes. The resulting deep red
solution was stirred at -78' for another 45 minutes. A
10 solution of 2-ada~aantanone (49.47 g, 0.33 mol) in 200 ml
TFiF was then added in a thin stream from the funnel over
5 minutes.
The cooling bath was removed and the stirred
mixture was slowly allowed to warm to approximately 0'
15 over 1.5 hours. At this point the slightly cloudy red
solution was heated to reflux for 4 hours whereupon a
clear, light red solution~was obtained after gas
evolution ceased.
During cooling to room temperature, the solution
20 became light yellow-brown after exposure to the
atmosphere. The mixture was carefully rotovapped
(foaming) to remove 750 ml of the solvent. Hexane (500
ml) was added, and the resulting slurry was extracted
with 500 ml water. The aqueous layer was back extracted
25 with hexane (250 ml) and the combined organics were
extracted with saturated brine (2~x 250 ml). The hexane
solution was dried over anhydrous potassium carbonate
and treated with 1 g. decolorizing carbon. Filtration
through celite, followed by evaporation produced a light
30 yellow viscous oil which was pumped at 90' with stirring
under high vacuum to remove a small amount of residual
adamantanone.
The final weight of the crude product was 94 g.
The infrared spectrum showed no carbonyl absorption due
35 to adamantanone (1705 cm~) or the corresponding
adamaatyl methoxyphenyl ketone (1670 cm'). l~lthough~
this product was sufficientllr pure far subseguent
. . ..._.,....~ 02340484 2001-03-19 . . . . .. .. ..
41
reaction, it was found that an identical procedure using
46.8 g. 2-adamantanone (0.9 equivalents) provided an
oil, which when passed through a silica gel column (15
cm x 3.5 cm) and eluting with 2% ethyl acetate in
hexanes, gave an oil which solidified in the cold.
Recrystallization from a minimal amount of hexanes
yielded a waxy, white solid melting at 34-37. C. Anal.
Calcd for C»FiZ,~Ot: C, 80.24: H, 8.51. Found: C, 81.23:
H, 8.49.
Both the crude oil and the waxy solid gave
identical I . R. and NI~t spectra
I.R. (neat, cm~); 2900, 283$, 2655, 2640, 2620,
1655, 1600, 1592, 1580, 1'574, 1444, 1282, 1240, 1202,
1095, 1078.
~HNI~t (400 I~iz, CDC13) : d 1.75 - 2.05 (12H, m,
adamantyl): 2.66 (1H, br s, Hay); 3.27 (1H, br s, Ha2);
3.31 (3H, s, OCH3); 3.83 (3H, s, ArOCH3): 6.82 - 6.94
(3H, m); 7.23 - 7.30 (1H, m).
~3
~3
Example 7
3-Pivaloyl~benzaldehyde
3-Hydroxybenzaldehyde (2.04 g., 16.7 mmol) in 25 ml
dichloromethane under an argon atmosphere was treated
with triethylamine (3.5 ml, 25.1 mmol). The solution
was cooled to 0' in an ice bath. Trimethylacetyl
chloride (2.3 ml, 18.4 nmol) was added dropwise via
syringe with magnetic stirring. After ten minutes, the
ice bath was removed and the mixture Yas stirred '-
overnight at room temperature. The reactiran was
....._ CA 02340484 2003-04-23 ..__.. ~.....a..,:;,-
;;....':~.:~....;.~'"";;:,.aLi.;_~.,'..~~...._...._,. ».._... -. ._ _ .. _.
.E;
42
~ quenched with 100 ml saturated sodium bicarbonate
solution. The organic layer was separated and the
aqueous layer extracted again with dichloromethane (2 x
30 ml). The combined organics were dried over innhydrous
sodium sulfate and concentrated ~ vacuo to an orange
residue which was passed through a short silica gel plug
with dichloromethane as eluent.
The solvent was evaporated from the silica gel
eluate to yield 3.40 g. (quant.j of the title compound
as a light yellow oil which was homogeneous according to
TLC (KSF, 20~ ethylacetate: hexanes). See Tables 3 and
? f or Hilt and IR data .
. fHfl
p
It
. G-C~t tCH3)3
The pivaloyl ester group is not deacylated under
the acidic conditions required for acetal and
phosphonate synthesis which are described in Examples 4
and 5 for the 3-methoxy derivatives, but they also serve
as general procedures. The resulting diethyl 1-methoxy-
1-(3-pivaloyloxyphenyljmethane phosphonate is used as
follows to procure methoxy (3-hydroxyphenyljmethylene
adamantine.
Lithium diisopropylamide ~LDA) solution was freshly r
prepared fn the following manner. A dry, three-necked, v.
2 Z, round bottomed flask Was equipped with a magnetic
stirring bar, a rellux condenser, a gas-inlet and a
500-ml dropping funnel. The flask and dropping funnel
were flamed in a stream of argon. To the flask eras
added 78 n1 (0.56 mole) of diisopragylamine and followed
by 500 ml of dry T~' ~Bal:es~reagent grade) . The
solution was stiarsed and cooled to -78° in an acetone-
... .._,..-? _ . .... ...._ :.:CA 02340484 2001-03-19 ... . . _.,. , . . . ..
... . . . .... . .... .. . . . .. _ . . . _.
43
dry ice bath, while 202 ml (0.51 mole) solution of 2.5 M
nbutyllithium in hexane (Aldrich) was transferred from
the bottle to a dropping funnel via a double-tipped
needle (3 ft., 16 gauge, Aldrich) and then added
dropwise to the solution over 20 min. After another 20
min. of stirring at -78', the 500-ml dropping funnel was
rapidly replaced with a 250-ml dropping funnel
containing a solution of 151.4 g (0.42 mole) of
phosphonate in 120 ml THF. The addition of phosphonate
to LDA solution at -78' caused a color change
immediately. After addition was completed (over 15
minutes), the resulting deep red mixture was stirred at
-78' for 1 hour longer. Then 49.1 g (0.33 mole) of
2-adamantanone was added. The mixture was stirred at
-78' for 10 minutes and allowed to warm to room
temperature in ca. 1.5 hour, and finally brought to
reflux for 1.5 hour. Vigorous gas evolution was noticed
during refluxing. The cooled reaction mixture was
treated with 0.5 L of saturated NaHC03 solution for 10
minutes and poured into a 4 L separatory funnel
containing 2 L of water. The aqueous phase Was extracted
trree times with 10~ EtOAc in hexane (3 x 250 ml). The
combined organic phase was washed with 1.5 L of water,
then with 1.5 L of brine and dried over Na2S0~. Removal
of solvent gave 135.5 g of viscous brown oil. The crude
product was diluted with 100 ml of 10~ EtOAc in hexane
and loaded onto a column (O. D.-4.5 cm., length-40 cm.),
packed with 80 g of silica gel (60-200 mesh, Baker).
Elution with 10 to 20~ EtOAc-hexanes gave five
fractions: 118 g of orange oil was recovered after
concentration, Which was a mixture of the pivaloyloxy
enol ether and the phenolic enol ether (Rf values. are
0.62 and 0.22, respectively, in 10~ EtOAc-hexanes) along
with in~puritfes. The oily pivaloyloxy enol ether was -
isolated by further chro~atography to provide an
analytical samples characterized by IR and ~HI~ (set
Tables 6 and 14~. -
CA 02340484 2001-03-19'
44
OL33,
O
0-C . C(Qi3)3
De-acylation of the mixture was completed in 2.5
hours by refluxing the mixture of crude products, 16.5 g
of KZC03 and 300 ml of MeOH. After removal of solvents
on a rotavap, an orange muddy solid was obtained. The
solid was treated with 200 ml of H20 and then scratched
vigorously with a spatula to afford a filterable
material. The solid was filtered and washed thoroughly
with 1.5 L of H20. After removal of most of the moisture
under vacuum, the slightly yellow solid was redissolved
in 600 ml of CHZC1Z (with gentle heating if necessary)
and dried over NaZSO~. The solution was filtered on a
Buchner funnel, packed with 40 g of silica gel. Upon
concentration to the half volume, a white solid began to
fall out of the solution. Recyrstallization in a
mixture of 1:1 CHZC12 and hexane gave 58.79 g (67%) of
white phenol enol ether (np: 131-133). Another 20-22 g
of product could be collected from the mother liquor
nfter chromatography.
OCEh
OH
CA 02340484 2001-03-19 - - .':: _:. ~., ~. _ .. _. .. ...... . . .
_:....._...... .. ..._ : .. .._.......,
Example 8
~-Acetoxybenzaldehyde
~3-Fiydroxybenzaldehyde (10 g., 81.88 mmol) was
dissolved in 150 ml dichloromethane under argon.
5 Triethylamine (17.12 ml, 0.123 mol) and dimethylamino-
pyridine (5 mg.) were added, and the resulting stirred
solution was treated with acetic anhydride (8.5 ml, 90
mmol). After stirring for fifteen hours, the reaction
mixture was transferred to a teparatory funnel using an
10 additional 50 ml dichloromethane. The organic layer was
washed with water (2 x 100 ml) and concentrated to give
a light brown oil weighing 14.85 g. Plug filtration
through silica gel using dichloromethane furnished
13.3 g (quant.) of a light,orange oil which was shown by
15 NMR and IR to be pure enough for use in subsequent
reactions (see Tables 3 and 7).
CHO
0 .
G-C-CN3
The aldehyde was converted to the corresponding
dimethyl acetal by way of the general procedure in
Example 4. The oily product, which was homogeneous
20 according to TLC, was obtained in good yield. The
structure was confirmed by proton NI~t and IR spectra
(see Tables 4 and 8). Conversion of the acetal to
diethyl 1-methoxy-1-(3-acetoxyphenyl) methane
phosphonate was carried out as in Example 5. N~t.and IR
25 spectral data confirmed the structure (sea Tables 5 and
9) and indicated that the crude product (oil) was pure
enough for subsequent use.
..... _ .._.,_ _~ 02340484 2001-03-19 '...._..._..._,..~.. ___,. . . ._. .
....~.__.... ._. ._.. ~.... .. ..
46
Example 9
Diethyl-1-methoxv-1-(3-hvdroxvohenyl)methang~~hosphonate
Diethyl-1-methoxy-1-(3-acetoxyphenyl)methanephos-
phonate from Example 8 (10.29 g., 32.56 mmol) :gas
dissolved in methanol (35 ml). Water (5 ml), and sodium
bicarbonate (5 g, 60 mmol) were then added with
stirring. After 48 hours at room temperature, the
reaction mixture was concentrated in vacuo to remove
methanol. The residue was treated with 150 ml
dichloromethane and washed with water (2 x 50 ml). The
organic layer was rotory evaporated and pumped at high
vacuum to yield 8.21 g. (93~) of the product as a light
yellow, viscous oil. Spectral data (Tables 5 and 9) are
.. in accordance with the structure:
CIi30 ~~ 0i t
~ of c
thoxvnaphthalene-lsarboxaldehvde dimet
6-Methoxynaphthalene-1-carbonitrile Was synthesized
from 6-methoxy-1-tetralone by the method of Harvey,
R.G., g~ ~, J Ora. Chem., 48:5134 (1983). The nitrile
(354.6 mg., 1.94 mmol) was dissolved in l0 ml dry
toluene under argon. The solution was cooled to -78' in
a dry ice/acetone bath. A toluene solution of DIBAL
(1.3 ml of a 1.5 M solution, 1.95 mmol) was added
dropwise by syringe with stirring. After 10 minutes the
mixture was warned slowly to room temperature and -
partitioned between 3N HCl'and dichloromethane (25 ml of
each). The organfc layer wa.s washed ~rith two additional
portions of 3N HCI. fhe combined aqueous layers were
CA 02340484 2001-03-19
47
back-extracted several times with 10 ml portions of
dichloromethane. The combined organics were dried over
Na2S04 and concentrated to yield yellow crystals of the
aldehyde which were immediately dissolved in methanol
(10 ml) and trimethyl orthofornate (0.25 ml, 2.29 mmol).
Several crystals of p-toluenesul-fonic acid were added,
and the solution was stored for 3 days in the
refrigerator. A small amount of NaHC03 was added and
the solvents were stripped. The residue was taken up in
to minimal dichloromethane and chromatographed on a silica
gel column using hexanes as the eluant. The appropriate
fractions were evaporated to furnish 395 mg. of the
title compound (88~ yield for 2 steps) as a light yellow
oil which was homogeneous on TLC and exhibited no
carbonyl absorption in the infrared spectrum. NI~t and
IR spectral data are consistent with the structural
assignment.
IR (CNC13, c~~): 2995, 2822, 1622, 1598, 1509,
1465, 1430, 1370, 1250, 1109, 1050, 841.
NI~t (CDC13, ppm): 3.36 (6N, s); 3.92 (3N, s); 5.85.
(IN, s); 7.16 (IN, d); 7.18 (IN, dd); 7.42 (IN, t); 7.56
(IN, d, J=7.08 Nz); 7.72 (1H, d, J=8.11 Hz); 8.19 (1H,
d, J=9.09).
Example lOB
Diethyl 1-methoxv-Z-l6-metho~y~aphth-1-yllmethane
pho~ohonate
The title phosphorate was synthesized according to
the general procedure described in Example 5. Spectral
data confirm the product structure.
IR (CNC13, c~ ~): 2994, 1619, 1594, 1504, 1458,
1429, 1372, 1242 (~O), 1050 (br), 968, 845, 810.
NI~t (CDC13, ppm) : 3.38 (3H, s) ; 3.92 (3H, s) ; 3.9 -
4.06 (4H, m); 5.25_(1H, d, J=16.4 HZ); 7.15 (1H, d, _
J=2.2 Hz): 7.18 (1H, dd, J=9.3, 2.85 Hz): 7.72 (1H, d,
J=8.05 Hz); 8.12 (11i, d, J=9.28 HZ).
CA 02340484 2001-03-19 .. . .. .... _. . .. . . : _ .... .. _.. ..._. .... . .
....... ._. . .
48
Example 11A
Methoxv-2-naphthaldeh~rde
6-Methoxy-2-naphthaldehyde was synthesized, using a
Bouveault reaction [E. A. Evans, J. Chem. Soc., 4691
(1956); P.T., Szzo, g~ ~, J. Ora. Chem., X4:701
(1959) ; D.C. Owsley, et ate, J-Org. Chem. , x:901
(1973)], by lithiating 5.08 g. (21.4 mmol) of 6-methoxy-
2-bromonaphthalene (dissolved in 50 ml dry THF) with
n-butylithium (13.7 m1, 21.8 mmol, 1.6 M) at -78' and
l0 quenching the arylilthium with dropwise addition of
sieve-dried dimethylformide (1.8 ml, 23.2 mmol). After
allowing the reaction to warm slowly to 0', the
intermediate aryl hydroxytamine was acidified with 3N
NC1 at 0'C, facilitating amine elimination to the
desired aldehyde. The solution Was partitioned between
EtOAc and 3N NC1, washing the aqueous layer 3 times with
EtOAc to recover all the aldehyde, and then the combined
EtOAc solutions were washed with saturated NaNC03
solution and dried over Na2S0~. After decanting and
evaporating the solution, the resultant oil was
dissolved in minimal CH2C12, followed by addition of
hexanes until the solution clouded. Refrigeration for
48 hours afforded 2.292 g (73~) of white crystals upon
filtration, which melted at 47-48'.
IR (CNC13, cm~~): 1685 (C=0),,1618, 1475, 1389,
1263, 1190, 1168, 1027, 895, 856, 839.
~H NMR (CDC13, ppm): 3.94 (3H, s); 7.16 (1H, d.
J=2.44 Hz): 7.21 (1H, dd, J=8.88, 2.44 Nz): 7.79 (1H, d,
J=8.55 Nz): 7.87 (1H, d, J=8.85 Hz): 7.90 (1H, d, J=8.55
Hz); 7.87 (1A, d, J=8.85 Hz): 7.90 (1H, dd, J=8.54, 1.52
Hz): 8.23 (1H, s): 10.07 (1H, s).
Examp 11B
6-Methoxv-2-naphthaldehyde dimethyl acetal -
The 6-methoxy-2-naphthyldimethyl acetal was
synthesized in 61~ yield (m. p. 27') according to the
procedure described in Example 4.
. . ..... _.... .,.._......._~.t.,._,......_.,.~ 02340484 2001-03-19~°'-
~~'~~-. ~~~.~ ~w.v--'mss'-~-:~::..~._____ . . .
49
IR (CNC13, cm~~): 2930, 2825, 1629, 1604, 1480,
1260, 1190, 1167, 1098, 1046, 890, 850.
~H NMR (CDC13, ppm): 3.38 (6H, s); 3.93 (3H, s):
5.54 (1H, s); 7.15 - 7.18 (2H, m); 7.52 (1H, dd, J=8.55,
1.51 Hz): 7.75 (1H, d, J=8.55 Hz); 7.76 (1H, d, J=8.55
Hz): 7.87 (1H, s).
Example 11C
Diethyl 1-Methoxv-1-l6-methoxvnapth-2-vllmethane
-- phosphate
The corresponding phosphonate was synthesized in
60% yield (oil) as described in Example 5.
IR (CNC13, cai~): 2998, 1619, 1603, 1480, 1390,
1258 (ps0), 1161, 1094, 1050 (br), 970, 852.
~H NMR (CDC13, ppm): 1.21 (3H, t, J=7.16 Hz): 1.26
(3H, t, J=7.16 Hz): 3.41 (3H, s); 3.92 (3H, s): 4.04 -
4.11 (4H, m): 4.64 (1H, d, J=15.41 Hz); 7.14 - 7.17 (2H,
m): 7.54 (1H, d, J=8.68 Hz): 7.75 (1H, d, J=8/86 Hz):
7.76 (1H, d, J=8.47 Hz): 7.82 (1H, s).
Example 11D
7-Methoxv-2-naphthaldehyde
7-Methoxy-2-naphthaldehyde was synthesized in 48%
yield (oil) using the Bouveault reaction as described
above.
IR (CNC13, Cm~): 1687 (C=0), 1601, 1460, 1389,
1331, 1266, 1175, 1115, 1030, 842.
. ~H NMR (CDC13, ppm) : 3.97 (3H, s) : 7.28 - 7.33 (2H,
m): 7.80 - 7.89 (3H, m): 8.25 (1H, s): 10.15 (1H, s).
Example 11E
7-Methoxv-2-naohthaldehvde dimethyl acetal
The corresponding dimethyl acetal was synthesized
in 86% yield (oil), following the conditions described'
in Example 4.
~H NMR (CDC13, pp~): 3.38 (6H, s): 3.93 (3H, s):
5.55 (lN,s~ ?.15 -:7.18 (2N, m): 7.42 (Ili, dd, J=8.03E
CA 02340484 2001-03-19 .--........._ . . ..
1.95 Hz) 7.75 (1H, d, J=9.77 Nz); 7.78 (1H, d, J=8.36
Hz): 7.85 (1H, s).
Example 11F
piet~yl 1-methox~y-1-l7-methoxvnap th-2~rllmethane
5 phosphonate
Diethyl 1-methoxy-1-(7-methoxynaphth-2-yl)methane
phosphonate was synthesized in 65% yield (oil) following
the general procedure outlined in Example 5.
IR (CNCl3, cm~~): 2295, 1630, 1603, 1460, 1390,
10 1256 (P=O), 1092, 1050 (br), 1027 (br), 970, 908, 840.
~H NMR (CDC13, ppm): 1.22 (3H, t, J=7 Hz); 1.27
(3NH t, J=7 Hz) 3.43 (3H, s) 3.93 (3H, s) ; 4.04 - 4.11
(4H, m) 4.66 (1H, d, J=15.6 Hz); 7.15 - 7.17 (2H, m);
7.43 (1H, d, J=7.93 Hz): 7.73 (1H, d); 7.78 (1H, d,
15 J=8.46 Nz); 7.82 (1H, s).
CA 02340484 2001-03-19
51
TABLE
Acetal (A) Phosphonate (H)
R;O OR-' R'O POIOEt>=
Xt / It wH Xt / I H
\z
R= R=
Enol Ether (C)
R30
/ t =AD
Xt (
\'
R=
Example R=. R'. X Yield ~'~
t (~Icltine
Point)
IZ OCH;. Et. 100 99 9p
H
13 Hr, CH;. 91 89 86
H
(99-IOI)
14 NO=, CH;. 100 93
H
15 OCOC(CH;)~ 85 83 66
CH;. H ;- l45"c
of 1 %
C
16 OAc, CH;. 97 94 795'c of
H 17C
(131-133j
17 OH. CH;. Poor Poor
H
I8 OCH;, CH;. 99 ~ 94 93_95
H
l34-37)
19 OCH;. 98 R' _ -EcOEtR3 = -EtOEt
-CH=CH=-, 64 61
H
ZO OCH;. 72 ' 41 98
CH=C6H~.
H
Z1 OCH;. CH;, 99 95
4-OEt (65-67) (79-81)
2~ OCH;. CH;. 93 84 6a
2-CH;
(84-86)
Z3 H. CH;. 4-OCH;99 83 9g
(77_78.51
Enol ether yields based on 2-.4damantanone
AD is adamantyl.
CA 02340484 2001-03-19
52
H H H H
H
o z z = = x
E Z l ~ ._
d V a o _
.-, m o
~ 0 0 0; o
_e
v -
a _
N N jV
L Z
m
a ~
K _
_ _ r
N
a E E ~ o. = H m
~;
Z Z' ~ r o0
_v v ~ ~ ... ,_
~ 'C ~ 'C ~ 'C
~
O~ H H
H
t'
N ~ 'v .-~ N
N s o~ wo o.
w a f~ n n n f~
U . n
~D
E 1~ n
_
a
r~n
t
C
wv ~ xx
_~
H ,~ ~"
U H
O p Z Z =
a I ( ..
O f'! N 00
,..~ G v1 v1
A N .. ? O
I~ U _
~
o
W
a V
p,
>
e
=~
2'
'
a
t-
t vi ui ~n i
a
n
Z ~ ~
.e Z x
A
~ ~ n
a
a ~~ o
G: N o
a
~'
1 ey..1
a
3
..' V ~ Z
a U (>
O ~ ~~
' Z Z Z z
U ~ ~' V
U
~ o
I ~~ ~
I I
I N
I
- II II II II II II
II II II II
aG cx oG iG a
~ >C 'o
_ C x
C X
a
a
E N H N N 00
K
N
w
CA 02340484 2001-03-19
53
..,
z
a z ..
a
V
' o
N
x
I I ~ I
.,,
~1 ri
N
N N x N
N N N N N N N = ~ N N N ~ N
N r
xzz zzx x~ rzxx~, z~ N x
E ... v~ 00 00 0o oo ,o .o ~ z .o
y r ,o ~
I~ ~ ~-. ~
H
_ _
~rZ.,.:~9 EWvw Ezi~ Wo'd'd~ E H~oWd~~'d
~ E
=oz==x= xzxacz a~z~xxxzx'
=xxx=z ~ ~
_ _ ~- - .-. .._
iv - ~ N N - - - - . .... ' .. - ...
r r r 0y0 v1 ~ 0v0 h o0 a - - ~
r ~ ~ h ~ ~
0 N o0 - ~O
00 ~ N 00e1 N ~ ~ O ~ f ~ ~O ?
Y1 a0 N 11 1 fV N ~'1 N1
N1 00 O~ O~
O N
a w r ~ .c t~ e~ r e-: t~ :
r~ r r: t~ 00 0o r o
~ ~ o
n
~
o n ~
U O x r r ,
.
.
r
r
a
w o =i
o U N H N VI H VIH
x z o
x x ~ Z
x
x
a
Q !-U = . . . ,
o . o o o
o .
o
E. w ~ U ~ 25 r
I
a ,.; .; ..; ,~.; ..; ..;..;
N
j
x z
_ .. _
Z U H VI VI H VI VIH
z x x z z z
_
N ~O
V 1
VY V1 V1 h h V1h
x
V
V
"' ~ V=O
'
Z
a O O O
~ O ~ c
x E ~ E E E E E
a
o.
A N O A1 A1 f1 A Aj
H
w
CA 02340484 2001-03-19
54
_ ~ N
~ Nx
i~
n
'~ f., H r Q' H H
_ . uiE _ ~ xN~x_
_NE E O
_ N h n
h ? - n n
.xt = I O k ~i = Y e~i vi
H v
x~ N
n
v E EN E
00 .~
n N ~ h N x x
... r~1 V' 'nf h N
N H H
~~x_~xx
1 W.
V1 00 ~7
N _ ~ QO O~ O~
In o0 N a ~0 h~ ~O ~O ~O h
N N
xx~~E
E ~ ~ dD n H H H
'~ rT.. x
V Ex E Eziv~~~~' E ~ V~ T Z x
~ O x T~==x=x=~=
1 a ~; r~; r1
o :. ~h~ ~aa~~~,
h p. N ~ O O N a0 Q N
a .O v0 t~ ~O f~ h h ~O h h o m
n n
a ~ o ~ ~ x a x u.i
H H H H H H
~xv0 ~
r' v
o ~ V
a r~ .~ .~ I
x ~
..,
.,~
Ow H H H a
H H VI
x ~i x ac
a x ~, ~s
I
~; ri ~; ri
I
x ~ ,p n n
x
a a a ~ x
I I I v x
U
L a
E .o h oo a
r~ r1 r1 Q,
n ~ v
W
CA 02340484 2001-03-19
N J i.
n n V .
<' < ~ a
V 1I ~ ~ I ~ Y
r
. .,. ~. r ~ r, H
s i
~h
.n
w
_~N, ~ C
N ~ N _ N ~ N ~. N = N N N N N = N A W
_ r ~ CC 4 ~ ~ ~.
CC ~ ~C r ~ x r 0, C ~ .0 ~ . ~ .C ~ ,
_ _ ee _. _ r: 7 i _ n 7 . C . ~ ~ r: . r: r ,
E E C 7 7 E 'O L L E ~ S 9 L 7 7 . 9 7 7 . ~ 7 'O
IV ~ H N
awi = .~ ~ L ? ev P /V I~ X X X ~ .:~ n W i~ s "F ~ ~ v'. _ ._
s ~n-e.I ft.ev~ocrvn C-wn ~-n.r.c:.rr C=
< ~fJ 1r fr .0 1~ 1~ r~ f~ Ir ~ Ir a0 CD r e~ r: t~ r 1~ r: r .O .c 1~ r: . P
eV
c r:
i
a. n . .a
r P
_ < c t r:
U
U
C=
v
E a E i E Y E .r a = a
~, ~ i . . i
P . _ v1 . N s ..: 1n
I .-:s -:=~' es r. ~;~ .:,~ ~ H__
W E
< I n r.
Z . (;
N_ ~
V1 . C
v, n . ~ s n V'
rv_ rv
~Y. C ' 3 n 3 . ~ rrn ~ 3 C:
E,., Y J U "~~ = s_ . _ . n _ ,o P . n
;, ~ I . . r. ~ . - = = ~ C '°.. ,
v: _
I ~.
C=
V
Y H . 1Y
n
° ~
I . . ~ ~ .,; ~, . '
' n
O ~ ~ A ~ ~ _N ~ L~
P o0 P at P . v0 C~ ~C
V . 9 9 7 'O
V ~ ~n '~" '~" p . ~ i ' n
I f f v1 ~O V H f f
Y d' V Y '1'
n
n
U
U
C I ~ <
C = Z O C C J
E c E E E E E
I
a
r ~ ~ ~ ~ .rr ~ ~ a
L LL
CA 02340484 2001-03-19
56
_N
w P a E
a
. r;
_
o
_ . ,
a
r
7
.
-
r
_
._ O . ~N~
. ~, a ~ ~ . a
N
,. =
r ~
r ~ H a $ ~ P
r P
~. ? T (~ ni ~ 1 .~.. P
a _, ~'i Y
v
rv . n
C
", ~:.
n . r
n . ,C
. P
. v. r.
t
.nn r.r.~ . ,r
C ~
rr ~ i -
;" 7
7 ... 7 ~ ~ H
v. N
c E c '' -
_~ ~
__
C ~ C v : . a0 P 3 H n
r P
~ C,
~G h .p .G r r:
P~ t~ ~G r
= n .
. r r
Y. P ~
N r
~
t P W
. n'!
L .C r _
~C r
r
G
.
'
C
____ T__ _C_
T . < C t . Y
CI
L ~ T
t -
.
_
. 0..
:. t ~
.. "Y,~ ~~ ~ f
. '~1 r
L ~_ _
v,f f
.
n
c
... ,T,
V
Z
V
C
r r: ,p n
'
3
y~ : . . . v _
_
' . i
.
C= z _ r : _
r r: ?
C
_
J
N = K 1I
Vi~ ~
~ (~l
N
y H r r.
ac x s 's
r~. n
V L
W
i i
r. v
;.r
~ .
'
... .. ~ ,.._
1A A .. n .~i
L ,e
_ N
A N
~
7 C~ .p N P P
'
'
'
(J C
7
9
O
v C
U a . .=,
00 < H V
f f Y f
U
n ~ C
J sC W
-C V U
U
r. C
U U ._ V ._': ._::
;J O U U
x H f H h
a
vT1 H E 'V ~1 ~ H
A H V1 Y1 H
It
CA 02340484 2001-03-19
57
T
in .
S
Y ni I P
ac . ~ ~ ~ - _ n
X
. .
N
N
Ii
.swr
N K _
N A r'. ~ N
~
_ ,e N
, s
, ~ c
. _ ~ E . r
C
c x
.
. a e
. n 7
p ae
R
X Z -
- r 7
_ - _
L
? -
- N
C ~
'C r N r ~ _ ? N
r ~ v
C
.:~ s ~ m ~ ' r m
r~ r ~'
.~ ~ a
e
. a .p .G
t N Z !V _ T t~
a ? - .O
.r..~
a .e r: .o r r~
r r~ .c n r:
1Y
V
L L
r, v . a =
L t L C N H
_ N
a -_
l .r~.. ~ ~ V r
Y ~V N N N
~. ~. r~. v w
_ L L
a =
1I II II Y
= L L L V ev n
G: .Lv <
C . r ~
r
a .
Y G,
n rv <'i N
C' N N
_ r
?~ '
z ~ ~ a .
c
_ ... .~ .~. ... G ~'
Z 2' C C C C X ~ 1 i
p v~
7 t h
N N N N a - _
T ~
1' i_V p
G ?
?
_
N
N
1 1 1
C r N ~ r,7 I
r r r r R R
a _ _ _ _ _I ~
n
I
_
I
r1 Y1
i
a -, ..,
~ 1I
Y
~~ .
.
r.1 r.
r
7 UI ~~ T 1
1
n N n _ _-
a c
~ ~ ~ ~
n m
N N
a ~,
U
U
-
C C
U
z a e. E E U C
X r:~
a
c
, r
n h
~
. v E ~
,
.o
L
CA 02340484 2001-03-19
58
E
z
r
_ s~ via o'=I
- a N s .n a _ '.
n . n n v r I
_ c E E
E
E c ~'
~' '-_
.
. . :., .=
:r . C
~ . P N
J
.
i. ,C _ . _ r
-_ ,e . ,e n
. .
t T _ N ? P
I~ ?
?
i 17C p~
0C t _
N N
a .C .C .C
L L r~
r 1~
v v
_ L L L
< -_
C N C r:,
PI
n
.
z
L
_ L L
o _a ~ , a
L ~ h
N N N
z
C
..
C
J ~ C c
1
CI; ~ N ~ N
z ~ i
Q t p< N
Z E G ?_ P N
_ _ N
_
E .
n
a - Z
a _ _ _
II Y
Y
C
ao m ao
a i a .~. ~ n
~_ II
v '~ I I I
U~_ II . I
i a
U
H
N ~
:J U-~ U
oc .I I I
at -_
0
a c .
o ~ o
CA 02340484 2001-03-19
59
TABLE 7
IR SPECTRA OF ALDEHTDES
(All IR Spectra (Tables 7-101 Arc 'r eat C.'nlcss Otherwise Indicated cm-1)
CHO
Example R= R.
O 29b.>~. 1745 (ester C=O). 1695 (aldehyde
C=O), 1598. 1582, 1475, 123<, lI3Z. 1105
- m-OCC(CH3)3
67 m-OAc 2836. 1760 (ester C=O). 1695 (aldehyde
C=01. ISQB. 1583. 1376. 11x5
CHO
XI
x1 OCH;
6>i 4-OCH:CH; 2980. 28.'.5, 274.1. 2720.
1675(C=0).
(CHC13) 1580. 1573, 1460. 1262.
1130. 1027, 800
69 S-CHO 3005. 2837, 2723: 1695(C=0).
(CHCI3) 1590. 1463. 1'_9S. 1055.
863
70 2-CH3 2995.2930.2830.2720. 1685(C=0)
IS90. 1580. 1465. 1257,
1093. 1010. 780
71 O 29 76. 2925. 3820. 27.2.
1690(C=0)
1590. 1460, 1''47(P=0),
5-CH(OCH;)P(OEc1_ IISS, 1040, 865
CA 02340484 2001-03-19
60
TABLE 8
IR SPECTRA OF ACETALS
CH:O OCH:
/ H
R
Example R=
72 m-i'i0= 3080. 2930. 28=5. 1610.1580. 15=5, (Ar.''0~~.
1345 (ArNO=). 1?05. 1105, 1055. 98.. 805. 738,
715
73 O 2980. 2824. ) 748. (ester C=O). 1606, 1588, 1478.
1235, ) 145. 1110. 1055. 770
m-0CC(CH3)3
74 m-OAc 2930, 2824, I760 (ester C=O), 1605, 1585. 1365,
1200. 1096. 1050
75 m-OH 3450(0H). ?93.. 28?4, 1598. 1586, )450, 1348,
I 190. 1098. 1050. 778
76 p-0Me 2942, 2930. 2820. )606. 1580. 1508. 1?45, 1168,.
1096. 1050, 820
OR' OR'
I ~ ~H
Example R3 OCH:
77 -CHZCH3 2985. 2940--S0, 2870. 1600. 1585. 1485, 1330,
1260, 1100, 1030-50. 890. 875. 695
78 -CHzCHZ- 2880, 2940-50, 1600. 1585. 1485. 1390. 1315,
1260. 1100. 1030-50, 965, 945. 695
79 -CHZC6Fi5 3080. 3060, 3020. 2940-S0, 1600. 1585, 1485.
1430. 1260, 1 100. 1030-50. 865.695
CH:O OCH3
~H
Xt I
\?
OCH;
80 4-OCH~CH; 2995, 2930. 2820. 1601, 1587, 1507, 1410.
- (CHC13) )255. 1157. 1133. 1096. 1043. 975, 860
81 5-CH(OMe)_ 2980. 2910. 28Ø 1597, 1460, 1285, 1 ) 90.
1155. 1055(6r), 985. 855. 790
82 2-CHi 2980. _'9.5. 2820. 1580. 1465. 1350. 1255,
1190. 1075. 1050. 975. 91 ~, 78'. 764
CA 02340484 2001-03-19
61
TABLE 9
IR SPECTRA OF PHOSPHONATES
CH30 PO(OEc),_
~H
R=
Ezample R=
83 m-NO, 2950. 1610. 1578. 1526.
(ArNOz). 1350
(ArN02), 1250(P=0),
1095. 1048, 1022,
_ . 970
84 O ~ 2970, t745(cstcr C=O).
1603. 1584, 1475.
l272. 1253(P=0), 1135.
m-0CC(CHjj31110. 1020,
85 m-0Ac 2974, 1758(estcr C=O).
1603. 1584, 1250
(P=O). 1300. 1095. 1048,
1020
86 m-OH 32Ø 3190(0H), 2890,
1598. 1585. 1452.
1235(P=0), 1095. 1050.
1020. 965
87 p-OMe 2970. 1603. 1578. 1505.
1250(P=0). 1090,
1050. 1025. 9 7 5
88 m-Hr 2975, 1588. 1568. 1470.
1252(P=0), 1098.
1050, 1024. 96R
89 m-O!~fe 2974, 1596. 1582. 1480.
1255(P=0). 1098.
1050. 1020. 9b5
CA 02340484 2001-03-19
61B
Table 9
IR Spectra of Phosphonates
R~() 1't)(UE~)2
~I1
(H:li~
Example R'
90 -CHzCH'3~ 2975, 2925, 2900, 1595, 1580,1485, 1435,
1365, 1315, 1255, (P=O),1100, 1040 (br),
965, 870, 695
91 -CH~CH3-'~ 2975,2925, 2900, 1595, 1580, 1485, 1435,
ET 1365, 1315, 1255, (P=O),1100, 1040 (br),
965, 790, 695
92 -CH2C6H5 2-975,2925, 2900, 1595, 1580, 1485,1435,
1365, 1315, 1255 (P=O), 1160, 1100 ,
1040 (br), 965, 790, 740, 695
Ctl~() 1'<)(()~:oZ
~It
()C11~
93 4-OCHzCH3 2970, 2922, 2815, 1598, 1585, 1509,
1413, 1387, 1255 (P=O), 1090, 1040 (br),
960, 870, 790, 750
O
94 s-cx(ocH,)pl(ost>, 2978, 2925, 2900, 2820, 1590, 1458, 1387,
1330, 1250 (P=0), 1160, 1100, 1040-50 (br),
960, 870, 785, 695
95 2-CH3 2978, 2925, 2900, 2823, 1580, 1465, 1328,
' 1255 (P=O), 1168, 1050, 1020, 967, 785, 752,
730
CA 02340484 2001-03-19
62
TABLE 10
IR SPECTRA OF E.'rOL ETHI=RS
OCH3
/ \
R
\
Ezample -i~= -
96 O 2900. 2838. 2658. 2640. 2624, 1748fester
C=O). 1655. 1600. 1575, 1475. 1270. 1110
m-OCC(CH;)3
97 p-01~1e 2905, 28.10. 2656. 2640. 2620, 1652. 1602,
(CHCI;) 1520, 1504. 1240. 1086. 1075. 848
98 m-Hr 2910. 28.60, 2655. 2640. 2625. 1650, 1587,
(CHCI;) 1555, 1465, 1445. 1268, 1095, 1080
99 m-0Mc 2900, 2838. 2655, 2640, 2620. 1655, 1600,
1592. 1580, 1574, 14.a-1, 1282, 1240, 1202;
1095. 10'8
OR3
\
R~
OCH~
100 -CHa_CH; 2900, 38:0. 2660. 2625. 1655, 1600.
1590, 1580, 1572. 1480. 1460, 1442. 1425,
1385, 1280, 1240, 1195, 1175, 1045. 890,
782
101 CHzCH= 2900, 28x0. 2660. 2642, 2625. 1655. 1600.
1590, 1580. 1572. 1480. 1460, 1441. 145.
OEt 1380. 1280. 1240, 1195. 1120, 1045, 890, 782
102 CHiC6H~ 2900. 2840. 2660, 2642, 2625, 1655, 1600.
1590. 1580, 1573, 1480, 1425. 1280, 1240,
CA 02340484 2001-03-19
63
TABLE 10-continued
IR SPECTRA OF ENOL ETHERS
1'195. 1045. 1035. 960. 905. 865. 782. 695
~H3
X~
Example X~ OCH3
103 -4-OCH;CH~ 2900.2840.2655.2640, I6~.,0. 1653. 1598,
(CHC13) 1577, 1505, 1460. 1442, I245. 1135, 1033,
915, 865. 820
2-CH3 2910, 2840, 2660, 2640, 2625, 1575, 1465,
(CHC13) 1305. 1253, 1125, 1080, 1030, 970, 955
OCH3 OCH3
v
OCH3
105 H 2910. 2840. 1660. 2642, 2624. 1660. 1590.
(CHCI;) 1578, 1460. 14-t0, 1338. 135, 1245, 1160,
IOv~. 1078. 8.sn
CA 02340484 2001-03-19
64
Example 106
Phos~horvlation of Enol Ether Phenol
OR3 ORS
T ---~ T
0 0 0
o_~_o_M+
OH
O-M
_ . a - b
The enol ether phenol a (R' _ methyl, T = adamant-2-
ylidene,from Example 7) was reacted with 2-chloro-2-oxo-
1,3,2-dioxaphospholane according to Thuong, N.T., et al.,
Bull, Soc. Chim. France, 2083 (1975) to give a cyclic
phosphate triester, which underwent ring opening with NaCN to
yield the 2-cyanoethyl diester salt. Ammonium hydroxide then
induced a facile ~i-elimination reaction to a filterable
sodium ammonium salt of b (75's yield from a), which was ion
exchanged to the disodium salt of b for 1HNMR (D~0,400 MHz); $
1.6 - 7.9 (12H, m); 2.44 (1H, s); 2.97 (1H, s); 3.22 (3H, s):
6.98 (1H, m, 7.52 Hz): 6.96 (1H, s): 7.05 (1H, m): 7.18 (1H,
dd, 7.62, 8.06 Hz). This same salt was obtained directly
using sodium methoxide to induce ~i-elimination.
CA 02340484 2001-03-19
Example 107
Photooxvoenation of an Enol Ether Phosphate
OCH; ~O~ , O-O
h~ I
ocH;
00 ~ o
II
O-P-OH ~ O- j -O-Na ~
O O_Na+
i+ -
10 . H .
wherein T = adamantane
a b
The sodium ammonium salt a was ion exchanged to the
15 monopyridinium salt. A 0.06 M solution of the latter salt was
photooxygenated in the presence of 02 and TPP at 5°C. (Slower
reaction rates and increased photolytic damage to the product
were experienced with the use of solid phase sensitizers such
as Sensitox S or methylene blue on silica gel). Purification
20 on a reversed phase NPLC column at pN 8.6 (NaZC03) and using
an acetonitrile-water gradient, followed by lyophilization,
provided 3-(2'-spiroadamantane)-4-methoxy-4-(3"-
phosphoroyloxy) phenyl-1,2-dioxetane(b) as a faintly yellow
solid, in 80% yield.
25 1HNMR (D20, 400 MHz) : 0.85 (1H, d) ; 1.13 (1H, d) ; 1.40
1.67 (10H, m): 2.13 (1H, s): 2.75 (1H, s): 3.10 (3H, s); 7.15
(2H, broad, featureless); 7.20 (1H, d, 7.81 Hz); 7.28 (1H,
dd, 7.81, 8.09 Hz).
The upfield doublets are characteristic of the beta
30 adamantine ring protons in the dioxetane, which are more
shielded by the proximate aromatic ring than in the enol
ether. The coalescence of the two aromatic proton
CA 02340484 2003-04-23
66
resonances into a broad peak et x.15 ppm mirrors similar
behavior in the ~=C spectrum (D=0/CDsOD) t two aromatic
carbon resonanc~es at 120.95 ppm and 122.10 ppm are
broad, low intensity peaks at 0'C, which sharpen and
become more intense at 40'C. This indicates restricted
rotation of the aromatic substituent, which may
introduce a conformational. component into the rate of
electron transfer decomposition of ttie anion to the
excited state ester. .
~,?f~111r1.
Diethyl i-methoxy-1-(3-pivaloyloxyphenyl)methane
phosphonate (65.s g, 0:184 mol.y, prepared as described
ill United States Patent Number 5,225,584 , wag
placed in a dry 1 liter fiask.under argon. Dry tetra-
hydrofuran (165 ml.) was added, followed by
2~adamantanone (24.8 g, 0.185 mol.). The solution was
' stirred to homogeneity and set aside. In a separate
500 :Z. fias~c, n-butyllithium (81 ml. of a ~ 2.5 M
solution in hexanes) was added from a dropping funnel to
Zo a solution of diisopropylamine (30 ml., 0.14 moi.) in
200 ml. of tetrahydrofuran, which had been cooled irr a
dry ice-acetone bath to ~78'C under an argon atmosphere
The resulting solution of lithium diisopropylamide was
stirred at low temperature.for another Z5 minutes~and
Z5 then cannulated with a double tipped needle into the
solution of pbospbonate as~d.Z-adauantanone which bad
also beers cooled to -7B'C. ,Lithium diisopropylamide was
then added dropWise, with vigorous stirring,~over a 1.5
hour period. The clear, light brown reaction mixture
3o was then stirred for an additional 3.0 minutes at low
temperature, warmed to room temperature, and then
refluxe~d for Z.5 hours under argon and cooled to room -
temperature. Tbia layer chromatography (TLC) of the
crude reaction mixture (Whatman K5Ft iQ~t ethyl aeeta~e-
CA 02340484 2001-03-19 ._..r......__~__..._.._____ ..... .. _._._
67
hexanes) displayed three U.V. absorbing spots; one at
the origin, one at Rf.28, and the mayor spot at Rf.7o.
The thus-obtained reaction mixture was treated with
several aliquots of pivaloyl chloride, with stirring for
several hours at room temperature between additions.
After a total of 4.75 ml. (38.5 mmol.) of the acid
chloride had been added, TLC showed that the spot at
81.28 had completely disappeared. Thus, the lithium salt
of methoxy(3-hydroxyphenyljmethylene adamantine present
in the reaction-mixture had been converted to the
correspond-ing pivaloate ester at Rf.70. Tetrahydrofuran
was then partially removed by distillation at
atmospheric pressure to obtain a thick slurry, which was
then partitioned between water and 10~ ethyl acetate-
hexanes. The aqueous layer was separated and washed
again three times with the same solvent. The combined
organics were then washed several times with a saturated
aqueous solution of sodium bicarbonate, dried over
sodium sulfate, and filtered to remove any particulates.
Concentration of the solution on a rotory evaporator ,
gave a thick slurry of crystalline product. The slurry
was diluted with hexanes, cooled to -20', and filtered.
The filter cake was washed under argon with hexanes
which had been cooled in a dry ice-acetone bath. The
orange-brown filtrate was concentrated to an oil, which
was dissolved in minimal hexanes, seeded with crop 1 and
cooled to yield a second crop of the product. The
mother liquors from this operation were then plug
chromatographed on 74 g. of silica gel, eluting with
hexanes to leave the origin material (residual
phosphonate ester and its decomposition products)
behind. A third crop of product could then be obtained
upon concentration of the eluant. The total yield of
methoxy(3-pivaloyloxyphenyl) inethylene adamantine was -
54.67 g (79~j, melting point 83-B5'. Spectral data (1R,
and ~HN~t) Were identical to those previously reported in
CA 02340484 2003-04-23
68
PCT WO 89/06226r see Example 59
Example 109
A flame-dried flask was charged with methoxy(3-
pivaloyloxyphenyl)methane phosphonate (5.01 g, 14.1 mmol.).
Anhydrous methanol (40 ml.) was added under argon. The
resulting suspension was stirred vigorously during the
dropwise addition of 4.37 M sodium methoxide in methanol
(3.25 ml., 14.'2 mmol.). The suspended solid dissolved during
this operation. After stirring the mixture for one hour at
room temperature, TLC (Whatman*KSF; 10% ethyl acetate-
hexanes) showed that a very faint trace of the starting
material remained (Rf.70). One drop of the sodium methoxide
solution was added to the clear solution, which was then
concentrated on a rotory evaporator (bath temperature 35°)
and then pumped in vacuo (1.0 mm. Hg) at 40° for 24 hours.
The resulting dry, white solid, sodium 3-(methoxytri-
cyclo[3.3.1.1''']dec-2-ylidenemethyl)phenoxide, weighed 4.1 g.
(quantitative yield). It was insoluble in dichloromethane,
and TLC of the supernate showed no evidence for the presence
of any phenolic impurities. A nujo~mull of the product
displayed an infrared spectrum which was devoid of OH stretch
absorbances between 3500 and 3300 cm'''. The phenolate salt did
not exhibit a melting point below 280°, but did darken
somewhat beginning at 170°. It was kept dry during all
subsequent manipulations, and stored in a dessicator over
Drierite.
IR (nujol mull): 1572, 1405, 1310, 1285, 1198, 1175,
1150, 1090, 988, 870, 800, 777 cm 1.
Example 110
Sodium 3-(methoxytricyclo[3.3.1.13r]dec-2-
ylidenemethyl)phenoxide (1.74 g., 6.0 mmol.) was added under
argon to l0 inl. of scrupulously dried dimethyl-
*trade-mark
CA 02340484 2001-03-19
69
formamide containing several drops of triethylamine. The
resulting slurry was vigorously swirled during the
addition of 2-chloro-2-oxo-1,3,2-dioxaphospholane (0.580
ml., 6.3 mmol.) over 25 minutes. The mixture thinned
considerably during this addition and over an additional
3.5 hours of vigorous stirring at room temperature. Ury
sodium cyanide (0.325 g. 6.6 mmol.) was then added, with
exclusion of moisture, and stirring was continued
overnight at room temperature to give an orange, cloudy
solution. The solvent was removed in vacuo (1.0 mm Hg) at
50° and the residue was rinsed twice with o-oxylenes to
further eliminate DMF.
The resulting brown foam was dissolved in 10
ml. of methanol prior to the dropwise addition of 4.37 M
sodium methoxide in methanol (1.30 ml., 5.7 mmol.). After
30 minutes, the solvent was removed on the rotory
evaporator and the residue was slurred in 5~ water/-
acetone (v/v) and filtered. The solid filter cake was
dissolved in water and subjected to reverse phase
chromatography (PLRP polystyrene preparative HPLC column,
using a water-acetonitrile gradient) to conveniently
isolate disodium 3- (methoxytricyclo [3 .3 .1.13''] dec-2-
ylidenemethyl)phenyl phosphate in good yield as a white
fluffy solid after lyophilization of the appropriate
fractions. The 1HNMR spectral data for the product were
identical to those reported previously.
Particular compounds of the invention include:
i) the compound of formula:
CA 02340484 2001-03-19
Ne0 TO(OFt~=
o~
ii) a compound having the formula:
OR 3 OR 3
( R10 ) 20P .
PO ( ORl ) 2
R2
wherein R1 is a lower alkyl group having up to 12 carbon
atoms: R2 is an ether (0R4) or a thioether (SR4) wherein R4
is a lower alkyl or aralkyl substituent having up to 20
carbon atoms, an acetoxy or pivaloyloxy group, a ,halogen
atom, a nitro group, a hydroxyl group, an amino group, a di(lower
alkyl) amino group wherein each lower alkyl substituent contains
up to 7 carbon atoms, or an NHSOZRS group wherein RS is tolyl or
trifluoromethyl; - R' is a lower alkyl, aralkyl, aryl or
heteroaralkyl group having up to 20 carbon atoms, a (lower
alkyl)-O-SiX3 group wherein the lower alkyl moiety contains up to
7 carbon atoms, and any X is methyl, benzyl, or t-butyl, a
hydroxy (lower) alkyl group having up to 6 carbon atoms, or an
amino (lower) alkyl or di(lower alkylamino) group wherein each
lower alkyl group contains up to 7 carbon atoms and
CA 02340484 2001-03-19
71
Y is phenyl, biphenyl, 9,10-dihydrophenanthryl, naphthyl,
anthryl, phenanthryl, pyrenyl, dibenzosuberyl, phthalyl
or derivatives thereof.
iii) a compound having the formula:
( OEt )2 ( OEt ) 2
wherein RZ is an ether (0R4) or a thioether (SR4) wherein
R4 is a lower alkyl or aralkyl substituent having up to 20
carbon atoms, an acetoxy or pivaloyloxy group, a halogen
atom, a nitro group, a hydroxyl group, an amino group, a
di(lower alkyl) amino group wherein each lower alkyl
substituent contains up to 6 carbon atoms or an NHSOzRs
group wherein RS is tolyl or trifluoromethyl.
The above discussion of this invention is directed
primarily to preferred embodiments and practices thereof.
It will be readily apparent to those skilled in this art
that further changes and modifications in the actual
implementation of the concepts described herein can
easily be made without departing from the spirit and
scope of the invention as defined by the following
claims.
