U.S. patent application number 11/827650 was filed with the patent office on 2008-07-24 for isotopically labeled alpha-keto acids and esters.
This patent application is currently assigned to Spectra Gases, Inc.. Invention is credited to Frank Elbert Anderson, Rodolfo Antonio Martinez, Mark Minton, Erick Gabriel Ortiz, Kenneth Edmund Tortolani.
Application Number | 20080177115 11/827650 |
Document ID | / |
Family ID | 38895950 |
Filed Date | 2008-07-24 |
United States Patent
Application |
20080177115 |
Kind Code |
A1 |
Martinez; Rodolfo Antonio ;
et al. |
July 24, 2008 |
Isotopically labeled alpha-keto acids and esters
Abstract
Isotopically labeled alpha-keto acids and esters are disclosed
herein. Also disclosed are methods of synthesizing isotopically
labeled alpha-keto acids and esters.
Inventors: |
Martinez; Rodolfo Antonio;
(Santa Fe, NM) ; Minton; Mark; (Linville, VA)
; Anderson; Frank Elbert; (Germantown, MD) ;
Ortiz; Erick Gabriel; (Rio Rancho, NM) ; Tortolani;
Kenneth Edmund; (Mt. Airy, MD) |
Correspondence
Address: |
LERNER, DAVID, LITTENBERG,;KRUMHOLZ & MENTLIK
600 SOUTH AVENUE WEST
WESTFIELD
NJ
07090
US
|
Assignee: |
Spectra Gases, Inc.
Branchburg
NJ
|
Family ID: |
38895950 |
Appl. No.: |
11/827650 |
Filed: |
July 11, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60830301 |
Jul 11, 2006 |
|
|
|
60851706 |
Oct 13, 2006 |
|
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Current U.S.
Class: |
570/189 |
Current CPC
Class: |
C07C 59/19 20130101;
C07B 59/001 20130101 |
Class at
Publication: |
570/189 |
International
Class: |
C07C 21/02 20060101
C07C021/02 |
Claims
1. An isotopically labeled compound of Formula (IIa), ##STR00043##
wherein: X is F, Cl, Br, I, MgF, MgCl, MgBr and MgI; each R and
each R.sup.1 may be the same or different and independently may
represent hydrogen, deuterium, tritium or a C.sub.1-C.sub.36
substituted or unsubstituted, saturated, or unsaturated, linear,
branched, cyclic, aromatic, or substituted aromatic group, wherein
R or R.sup.1 may include a heteroatom including O, N, S, Si, and P
wherein any of the carbon atoms or heteroatoms may be isotopically
labeled; and y is independently 12, 13, or 14.
2. The isotopically labeled compound of claim 1, wherein at least
one of said .sup.yC is selected from the group consisting of
.sup.13C or .sup.14C.
3. The isotopically labeled compound of claim 1, wherein all of
said .sup.yC are .sup.12C.
4. The isotopically labeled compound of claim 1, wherein each R and
each R.sup.1 are independently selected from the group consisting
of hydrogen, deuterium, and a C.sub.1-C.sub.4 straight chain or
branched alkyl.
5. The isotopically labeled compound of claim 4, wherein each R and
each R.sup.1 are hydrogen.
6. The isotopically labeled compound of claim 5, wherein at least
one of said .sup.yC is selected from the group consisting of
.sup.13C or .sup.14C.
7. The isotopically labeled compound of claim 1, wherein X is
MgX.
8. The isotopically labeled compound of claim 7, wherein each R and
each R.sup.1 are independently selected from the group consisting
of hydrogen, deuterium, and a C.sub.1-C.sub.4 straight chain or
branched alkyl.
9. The isotopically labeled compound of claim 8, wherein each R and
each R.sup.1 are hydrogen.
10. The isotopically labeled compound of claim 9, wherein at least
one of said .sup.yC is selected from the group consisting of
.sup.13C or .sup.14C.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of the filing date of
U.S. Provisional Patent Application No. 60/830,301 filed Jul. 11,
2006 and U.S. Provisional Patent Application No. 60/851,706 filed
on Oct. 13, 2006, the disclosures of which are hereby incorporated
herein by reference.
BACKGROUND OF THE INVENTION
[0002] Pyruvic acid (C.sub.3H.sub.4O.sub.3) has the chemical name
2-oxopropanoic acid, and has a molecular mass of 88.06 grams per
mol.
[0003] Pyruvic acid is a colorless organic liquid formed as an
intermediate in carbohydrate metabolism and as an end product in
glycolysis. Pyruvic acid has a melting point ranging from about
11.degree. C. to about 12.degree. C. and is soluble in water.
[0004] In the laboratory, pyruvic acid may be prepared by heating a
mixture of tartaric acid and potassium hydrogen sulfate, or by the
hydrolysis of acetyl cyanide, formed by a reaction of acetyl
chloride with potassium cyanide. Production under these conditions,
however, leaves undesirable impurities, which can be toxic or
harmful if not removed in entirety.
[0005] Pyruvic acid also occurs naturally as an intermediate
product in carbohydrate and protein metabolism in the human body.
Pyruvic acid is important in metabolism as it can be converted to
carbohydrates via gluconeogenesis, to fatty acids or energy through
acetyl-CoA (which is the main input for a series of reactions known
as the Krebs cycle), to the amino acid alanine, and to ethanol.
[0006] In industry, pyruvic acid is used to produce its salts and
esters (pyruvates) for the use as dietary supplements and as an
effective means of weight loss. Pyruvic acid is also used for the
synthesis of amino acids and used for biomedical research. Its
derivatives are used in making food additives and flavoring
agents.
[0007] Unfortunately, due to the highly reactive nature of pyruvic
acid, storage of the molecule over extended periods of time is very
difficult and undesirable.
[0008] U.S. Pat. No. 6,753,446 describes diethyl oxalate analogs
useful for asymmetric labeling of synthetic compounds. The
compounds have the general structure RO--C(O)C(O)--X
[0009] United States Patent Publication No. 2007/0106085 describes
intermediates useful in the preparation of
[.sup.13C.sub.1-5]metacrylic acid.
[0010] United States Patent Publication No. 2006/0178534 describes
labeled compounds useful for the preparation of labeled compounds,
including pyruvic acid.
SUMMARY OF THE INVENTION
[0011] In accordance with one embodiment of the present invention
there is provided an isotopically labeled compound of Formula
(IIa),
##STR00001##
wherein:
[0012] X is F, Cl, Br, I, MgF, MgCl, MgBr and MgI;
[0013] each R and each R.sup.1 may be the same or different and
independently may represent hydrogen, deuterium, tritium or a
C.sub.1-C.sub.36 substituted or unsubstituted, saturated, or
unsaturated, linear, branched, cyclic, aromatic, or substituted
aromatic group, wherein R or R.sup.1 may include a heteroatom
including O, N, S, Si, and P wherein any of the carbon atoms or
heteroatoms may be isotopically labeled; and
[0014] y is independently 12, 13, or 14.
[0015] In accordance with another embodiment of this invention,
each R and each R.sup.1 are independently selected from the group
consisting of hydrogen, deuterium, and a C.sub.1-C.sub.4 straight
chain or branched alkyl.
[0016] Recently, isotopically enriched pyruvic acid was studied for
its use in a variety of medical diagnostic applications. The use of
pyruvic acid that is isotopically enriched with at least one carbon
13 isotope allows for its use in medical diagnostics. Due to the
highly reactive nature of pyruvic acid, storage of the molecule
over extended periods of time is difficult and undesirable.
Fortunately, storage of pyruvic acid is possible by forming shelf
stable precursors of pyruvic acid, and then converting the
precursor to pyruvic acid prior to its use, for example, as a
medical diagnostic agent.
[0017] Disclosed are two synthetic pathways for the synthesis of
precursors of pyruvic acid. The synthesis described herein provides
pyruvic acid precursors of the present invention and derivatives in
high yield which are shelf stable and which are of a high purity.
Moreover, the pyruvic acid precursors allow conversion to pyruvic
acid in one step. Finally, the synthetic methodologies of the
present invention avoid the use of dangerous reagents, such as
potassium cyanide.
DETAILED DESCRIPTION
[0018] The present invention provides compounds having Formula
(I)
##STR00002##
wherein: G represents a halogen or a Grignard-halogen complex such
as MgF, MgBr, MgCl, and MgI;
##STR00003##
[0019] wherein .sup.+A represents a positively charged
counterion;
[0020] Q represents C or O, each of which may be isotopically
labeled;
[0021] Q' represents O or N, each of which may be isotopically
labeled;
[0022] each R, each R.sup.1, and each R.sup.2 may be the same or
different and independently may represent hydrogen, deuterium,
tritium or a C.sub.1-C.sub.36 substituted or unsubstituted,
saturated, or unsaturated, linear, branched, cyclic, aromatic, or
substituted aromatic group, wherein R or R.sup.1 may include a
heteroatom including O, N, S, Si, and P wherein any of the carbon
atoms or heteroatoms may be isotopically labeled;
[0023] m is 1 if Q' is O or 2 if Q' is N;
[0024] n is 0 if Q is O or 2 if Q is C; and
[0025] y is independently 12, 13, or 14;
[0026] wherein the compound is not unlabeled pyruvic acid, the
salts of pyruvic acid, unlabeled benzyl pyruvate, unlabeled benzyl
methacrolate, propanoic-3-.sup.13C acid-2-oxo-phenylmethyl ester;
or 2-propenoic acid-2-(methyl-.sup.13C-d3)-phenylmethyl ester.
[0027] As used herein, the terms "isotope", "isotopic" or
"isotopically labeled" refer to an atom having the same number of
protons but a different number of neutrons as compared with the
most abundant form of the element. Accordingly, carbon may be
isotopically labeled as .sup.13C, nitrogen may be isotopically
labeled as .sup.15N, sulfur may be isotopically labeled as .sup.32S
and oxygen may be isotopically labeled as .sup.16O, .sup.17O or
.sup.18O. These terms as used herein also refer to radio-labeled
elements. Further, these terms as used herein also refer to
molecules which contain isotopic atoms.
[0028] The terms "aromatic" or "cyclic group" as used herein,
encompasses not only the group but also the substitutions in one or
more positions. Substitutions may include, and without limitation,
halogens, hydroxyl, nitro, amino, substituted amino having the
formula --N(R.sup.3)(R.sup.3), (wherein R.sup.3 is a
C.sub.1-C.sub.6 linear, branched, or cyclic alkyl group),
C.sub.1-C.sub.5 alkoxy, or C.sub.1-C.sub.5 alkyl groups. Thus, for
example, a reference to a benzyl group can include, for example,
meta-chloro benzene, 3,4,5 tri-bromo benzene, p,m,o-methyl,
p,m,o-methoxy, and trifluoromethyl.
[0029] Hydrogen atoms, which by convention are not shown, may be
deuterium or tritium.
[0030] In a preferred embodiment, at least one atom in Formula (I)
is isotopic. More preferably, at least one carbon or .sup.yC is
C.sup.13 or C.sup.14.
[0031] The present invention also provides compounds of Formula
(II)
##STR00004##
wherein:
[0032] X represents F, Cl, Br, I, MgF, MgCl, MgBr and MgI;
[0033] Q represents C which may be isotopically labeled;
[0034] each R, each R.sup.1, and each R.sup.2 may be the same or
different and independently may represent hydrogen, deuterium,
tritium or a C.sub.1-C.sub.36 substituted or unsubstituted,
saturated, or unsaturated, linear, branched, cyclic, aromatic, or
substituted aromatic group, wherein R or R.sup.1 may include a
heteroatom including O, N, S, Si, and P wherein any of the carbon
atoms or heteroatoms may be isotopically labeled;
[0035] n is 2; and
[0036] y is independently 12, 13, or 14.
[0037] In some embodiments, each R and each R.sup.1 each may be the
same or different and independently may be selected from hydrogen,
deuterium, tritium or a C.sub.1-C.sub.24 substituted or
unsubstituted, saturated or unsaturated, linear, branched, cyclic,
aromatic or substituted aromatic moiety optionally containing one
or more heteroatoms including O, N, S, P, and Si, any of which may
be isotopically labeled.
[0038] In other embodiments, each R and each R.sup.1 may be the
same or different and independently may be selected from hydrogen,
deuterium, tritium or a C.sub.1-C.sub.16 substituted or
unsubstituted, saturated or unsaturated, linear, branched, cyclic,
aromatic or substituted aromatic moiety optionally containing one
or more heteroatoms including O, N, S, P, and Si, any of which may
be isotopically labeled.
[0039] In yet other embodiments, each R and each R.sup.1 may be the
same or different and independently may be selected from hydrogen,
deuterium, tritium or a C.sub.1 to C.sub.6 linear or branched,
substituted or unsubstituted, cyclic, or aromatic moiety,
optionally containing one or more heteroatoms including O, N, S, P,
and Si, any of which may be isotopically labeled.
[0040] In preferred embodiments of the compositions of Formula
(II), each R, each R.sup.1, and each R.sup.2 may be the same or
different and independently may be selected from hydrogen,
deuterium, methyl, ethyl, propyl, isopropyl, butyl, secbutyl,
tertbuytl, allyl, 2-butenyl, 3-butenyl, phenyl, benzyl, napthyl,
cyclopropyl, cyclopentyl, and cyclohexyl, thienyl, furyl, pyridyl,
imidazoylyl, benzimidazoyl, or benzothiazolyl.
[0041] In a preferred embodiment, at least one atom in Formula (II)
is isotopic. More preferably, at least one carbon is .sup.13C or
.sup.14C. Most preferably, at least one .sup.yC is .sup.13C or
.sup.14C.
[0042] In some embodiments, the compounds of Formula (II) have the
structure of Formula (IIa):
##STR00005##
wherein R and R.sup.1 are as defined previously.
[0043] In other embodiments, the compounds of Formula (II) have the
structure of Formula (IIb):
##STR00006##
wherein R and R.sup.1 are as defined previously.
[0044] Non-limiting examples of compounds falling within the scope
of the compounds of Formula (II) include:
##STR00007##
[0045] The present invention also provides compounds of Formula
(III)
##STR00008##
wherein:
[0046] Q represents C or O, each of which may be isotopically
labeled;
[0047] Q' represents O, or N, each of which may be isotopically
labeled;
[0048] each R, each R.sup.1, and each R.sup.2 may be the same or
different and independently may represent hydrogen, deuterium,
tritium or a C.sub.1-C.sub.36 substituted or unsubstituted,
saturated, or unsaturated, linear, branched, cyclic, aromatic, or
substituted aromatic group, wherein R or R.sup.1 may include a
heteroatom including O, N, and S, wherein any of the carbon atoms
or heteroatoms may be isotopically labeled;
[0049] m is 1 or 2;
[0050] n is 0 or 2; and
[0051] y is independently 12, 13, or 14;
[0052] wherein the compound is not unlabeled pyruvic acid, the
salts of pyruvic acid, unlabeled benzyl pyruvate, unlabeled benzyl
methacrolate, propanoic-3-.sup.13C acid-2-oxo-phenylmethyl ester;
or 2-propenoic acid-2-(methyl-.sup.13C-d3)-phenylmethyl ester.
[0053] In a preferred embodiment, at least one atom in Formula
(III) is isotopic. More preferably, at least one carbon or .sup.yC
is C.sup.13 or C.sup.14
[0054] In some embodiments, each R, each R.sup.1, and each R.sup.2
may be the same or different and independently may be selected from
hydrogen, deuterium, tritium or a C.sub.1-C.sub.24 substituted or
unsubstituted, saturated or unsaturated, linear, branched, cyclic,
aromatic or substituted aromatic moiety optionally containing one
or more heteroatoms including O, N, S, Si, and P any of which may
be isotopically labeled.
[0055] In other embodiments, each R, each R.sup.1, and each R.sup.2
may be the same or different and independently may be selected from
hydrogen, deuterium, tritium or a C.sub.1-C.sub.16 substituted or
unsubstituted, saturated or unsaturated, linear, branched, cyclic,
aromatic or substituted aromatic moiety optionally containing one
or more heteroatoms including O, N, and S, any of which may be
isotopically labeled.
[0056] In yet other embodiments, each R, each R.sup.1, and each
R.sup.2 may be the same or different and independently may be
selected from hydrogen, deuterium, methyl, ethyl, propyl,
isopropyl, butyl, secbutyl, tertbuytl, allyl, 2-butenyl, 3-butenyl,
phenyl, benzyl, napthyl, cyclopropyl, cyclopentyl, cyclohexyl,
thienyl, furyl, pyridyl, imidazoylyl, benzimidazoyl, or
benzothiazolyl.
[0057] In yet further embodiments, R.sup.2 is selected from a
protecting group including but not limited to methoxymethyl ether,
tetrahydropyranyl ether, t-Butyl ether, allyl ether, benzyl ether,
trimethylsilyl ethers, triethylsilyl ethers, t-butyldimethylsilyl
ether, t-butyldiphenylsilyl ether, acetic acid ester, benzoic acid
ester, methylthiomethyl ethers, benzyloxymethyl ethers,
2-napthylmethyl ethers, p-methoxybenzyl ethers, trityl ethers, and
methoxytrityl ethers.
[0058] In some embodiments, Q is C; Q' is O; and R.sup.2 is H, and
at least one .sup.yC group is .sup.13C or .sup.14C.
[0059] In other embodiments, Q is C; Q' is O; and R.sup.2 is
benzyl, and at least one .sup.yC group is .sup.13C or .sup.14C.
[0060] In further embodiments, Q is O; Q' is O; and R.sup.2 is
benzyl, and at least one .sup.yC group is .sup.13C or .sup.14C.
[0061] In yet other embodiments, the compounds of Formula (III)
have the structure of Formula (IIIa):
##STR00009##
wherein .sup.yC, R, R.sup.1, and R.sup.2 are as defined
previously.
[0062] In yet further embodiments, the compounds of Formula (III)
have the structure of Formula (IIIb):
##STR00010##
wherein .sup.yC, R, R.sup.1, and R.sup.2 are as defined
previously.
[0063] In preferred embodiments, the compounds of Formula (III)
have the structure of Formula (IIIc)
##STR00011##
wherein:
[0064] Q, .sup.yC, R, and R.sup.1 are as defined previously;
and
[0065] R.sup.4 represents a C.sub.1-C.sub.10 aromatic ring
optionally substituted with one or more nitro, amino, substituted
amino having the formula --N(R.sup.3)(R.sup.3), halogen, deuterium,
tritium, C.sub.1-C.sub.4 alkoxy, or C.sub.1-C.sub.4 alkyl groups,
wherein the aromatic ring optionally includes a heteroatom selected
from the group consisting of O, N, and S.
[0066] Examples of aromatic rings representative of R.sup.4
include, but are not limited to, phenyl, napthyl, benzofuran,
isobenzofuran, indole, benzothiophenee, benzimidazole, indazole,
benzoxazole, benzisoxazole, benzothiazole, pyridine, quinoline,
isoquinoline, pyrazine, quinoxaline, pyridazine, cinnoline and the
substituted variants thereof.
[0067] It has been found that the inclusion of a single carbon
spacer adjacent to R.sup.4, as in Formula (IIIc) and the Formulas
which follow, allows for cleavage of the R.sup.2 group (Formula
(III)) by means of hydrogenation rather than acid hydrolysis. As
discussed herein, hydrogenation provides an efficient means by
which the compounds of Formula (IIIc) can eventually be converted
to pyruvic acid or its derivatives thereof. Moreover, hydrogenation
allows for the production of pyruvic acid under neutral conditions
and where the only byproduct is toluene. Other methods, including
acid hydrolysis, leave inorganic or organic acids as
impurities.
[0068] In more preferred embodiments, the compounds of Formula
(III) have the structure of Formula (IIId):
##STR00012##
wherein Q, .sup.yC, R, and R.sup.1 each are as defined previously;
and R.sup.5 represents at most 5 substitutions on the aromatic
ring, wherein R.sup.5 independently represents nitro, amino,
substituted amino having the formula --N(R.sup.3)(R.sup.3),
halogen, deuterium, tritium, C.sub.1-C.sub.4 alkoxy, or
C.sub.1-C.sub.4 alkyl group.
[0069] In one particularly preferred embodiment, the compounds of
Formula (IIId) have the structure of Formula (IIIe):
##STR00013##
wherein .sup.yC, R, R.sup.1, and R.sup.5 are as defined
previously.
[0070] In another particularly preferred embodiment, the compounds
of Formula (IIId) have the structure of Formula (IIIf):
##STR00014##
wherein .sup.yC, R.sup.1, and R.sup.5 are as defined
previously.
[0071] In another particularly preferred embodiment, the compounds
of Formula (IIIe) have the structure of Formula (IIIg):
##STR00015##
wherein .sup.yC, R, and R.sup.5 are as defined previously.
[0072] In another particularly preferred embodiment, the compounds
of Formula (IIIe) have the structure of Formula (IIIh):
##STR00016##
wherein .sup.yC and R.sup.5 are as defined previously.
[0073] In yet another particularly preferred embodiment, the
compounds of Formula (IIIf) have the structure of Formula
(IIIi):
##STR00017##
wherein .sup.yC and R.sup.5 are as defined previously.
[0074] In a most preferred embodiment of Formula (IIIi), each
R.sup.5 is hydrogen.
[0075] In yet another particularly preferred embodiment, the
compounds of Formula (IIId) have the structure of Formula
(IIIj):
##STR00018##
wherein .sup.yC, Q, n, R, R.sup.1, and R.sup.5 are as defined
previously.
[0076] As illustrated below in Formula (IIIj), in a preferred
embodiment in accordance with the present invention and
particularly for those isotopically labeled compounds discussed
herein having the structures generally shown in Formulas IIIa
through IIIi, as well as Formula V, it is preferred that the double
bond oxygen on the ester forming carbonyl, the carbon of the ester
forming carbonyl and/or the carbon of the methacrylate or ketone
group be isotopically labeled. In a particularly preferred
embodiment in accordance with the present invention, the isotopic
labeling of the compounds described above would occur at some atom
other than the carbon bound to the various R.sup.1 groups.
Isotopically labeling may occur at a plurality of other groups as
well.
[0077] Non-limiting examples of the compounds of Formulas (IIIe)
and Formula (IIIf) include:
##STR00019## ##STR00020##
[0078] In other embodiments, the compounds of Formula (I) have the
structure of Formula (IV):
##STR00021##
wherein .sup.yC, Q, R, and R.sup.1 are as defined previously.
[0079] Non-limiting examples of the compounds of Formulas (IV)
include:
##STR00022##
wherein R represents R.sup.2 of Formula (IV).
[0080] In the propamides of Formula (IV) above, R is preferably a
C.sub.1-C.sub.4 alkyl, more preferably methyl or ethyl.
[0081] The present invention also provides a method of synthesizing
isotopically labeled compounds including analogs of pyruvic
acid.
[0082] One synthetic method, according to the following scheme,
comprises reacting a compound of Formula (IIa) with magnesium
turnings in a solvent to yield a compound having Formula (IIb):
##STR00023##
[0083] Preferably, the solvent is an aprotic solvent. More
preferably, the solvent is an ether or toluene.
[0084] Preferably, the reaction is run at room temperature, more
preferably the reaction is initially run at room temperature with a
subsequent increase in temperature to drive the reaction to
completion. As used herein, "room temperature" means a temperature
ranging from about 22.degree. C. to about 26.degree. C.
[0085] In a preferred embodiment, each R and each R.sup.1 may be
the same or different and independently may be selected from
hydrogen or C.sub.1-C.sub.4 alkyl, more preferably each R and each
R.sup.1 are hydrogen.
[0086] Another synthetic method, according to the following scheme,
comprises reacting a compound of Formula (IIb)) with labeled or
unlabeled carbon dioxide in a solvent to yield a compound having
Formula (IIIa'):
##STR00024##
[0087] Preferably, the solvent is selected from ether,
tetrahydrofuran, dioxane, and glymes.
[0088] Preferably, the reaction is run with cooling, more
preferably at a temperature of about 0.degree. C. or below, most
preferably at about -50.degree. C. or below.
[0089] In a preferred embodiment, each R and each R.sup.1 may be
the same or different and independently may be selected from
hydrogen and C.sub.1-C.sub.4 alkyl, more preferably each R and each
R.sup.1 are hydrogen.
[0090] Another synthetic method, according to the following scheme,
comprises reacting a compound of Formula (IIIa') with a weak base
in the presence of a reagent having a halogenated leaving group to
yield a compound having Formula (IIIb):
##STR00025##
[0091] Preferably, bases include K.sub.2CO.sub.3, NaHCO.sub.3,
Li.sub.2CO.sub.3, Cs.sub.2CO.sub.3, t-ButOK, t-ButOLi, hydroxides,
alkyl salts, lithium salts, metal hydrides, and heteroatom bases.
More preferably the base is K.sub.2CO.sub.3.
[0092] In a preferred embodiment, R.sup.2X is benzyl chloride or
benzyl bromide.
[0093] In a preferred embodiment, the reaction is run at room
temperature or below. One skilled in the art would recognize that
the reaction may be run at temperatures lower than room temperature
(between room temperature and -78.degree. C.) to accommodate
certain bases.
[0094] In a preferred embodiment, each R and each R.sup.1 may be
the same or different and independently may be selected from
hydrogen and C.sub.1-C.sub.4 alkyl, more preferably each R and each
R.sup.4 are hydrogen.
[0095] In a preferred embodiment R.sup.2 is --CH.sub.2--R.sup.4,
wherein R.sup.4 includes, but is not limited, to phenyl, napthyl,
benzofuran, isobenzofuran, indole, benzothiophenee, benzimidazole,
indazole, benzoxazole, benzisoxazole, benzothiazole, pyridine,
quinoline, isoquinoline, pyrazine, quinoxaline, pyridazine,
cinnoline and the substituted variants thereof.
[0096] Yet another synthetic method, according to the following
scheme, comprises reacting a compound for Formula (IIIa) with ozone
in a solvent to yield a compound having Formula (IIIb):
##STR00026##
[0097] Preferably, this reaction is run in a C.sub.1-C.sub.6
alcohol (straight chain or branched), methylene chloride,
chloroform and the like.
[0098] Preferably, the reaction is run at a reduced temperature,
more preferably at about 0.degree. C. or below. As used herein, the
term "reduced temperature" refers to a temperature below room
temperature.
[0099] In a preferred embodiment, each R and each R.sup.1 may be
the same or different and independently may be selected from
hydrogen and C.sub.1-C.sub.4 alkyl, more preferably each R and each
R.sup.1 are hydrogen.
[0100] In a preferred embodiment R.sup.2 is --CH.sub.2--R.sup.4,
wherein R.sup.4 includes but is not limited to, phenyl, napthyl,
benzofuran, isobenzofuran, indole, benzothiophenee, benzimidazole,
indazole, benzoxazole, benzisoxazole, benzothiazole, pyridine,
quinoline, isoquinoline, pyrazine, quinoxaline, pyridazine,
cinnoline and the substituted variants thereof. In a more preferred
embodiment, R.sup.2 is benzyl.
[0101] Yet a further synthetic method, according to the following
scheme, comprises converting a compound having Formula (IIIb) to
isotopically labeled pyruvic acid or an analog thereof by means of
hydrogenation, preferably in a solvent selected from a
C.sub.1-C.sub.6 alcohol (straight chain or branched), a ketone,
water, an ether, or mixtures thereof.
##STR00027##
[0102] The reaction is preferably run under a pressure of about 4
to about 12 psig, more preferably the reaction is run using a
palladium catalyst, even more preferably the palladium catalyst is
on charcoal.
[0103] The reaction is preferably run at room temperature.
[0104] In a preferred embodiment, each R.sup.1 is independently
selected from hydrogen or C.sub.1-C.sub.4 alkyl, more preferably
each R.sup.1 is hydrogen.
[0105] The above conversion can also be carried out via acid
hydrolysis using techniques known to those of skill in the art.
United States Patent Publication No. 2006/0178534, incorporated
herein by reference, describes a method of performing acid
hydrolysis. For example, acid hydrolysis may be accomplished by
treating a compound with 1M HCl and then extracting the final
product with an organic solvent, preferably ethyl acetate.
[0106] Yet a further synthetic method, according to the following
scheme, comprises converting a compound having Formula (IIIf) to
isotopically labeled pyruvic acid or an analog thereof by means of
hydrogenation, preferably in a solvent selected from a
C.sub.1-C.sub.6 alcohol (straight chain or branched), a ketone,
water, an ether, or mixtures thereof.
##STR00028##
[0107] The reaction is preferably run under a pressure of about 4
to about 12 psig, more preferably run using a palladium catalyst,
even more preferably the palladium is on charcoal.
[0108] In a preferred embodiment, each R.sup.1 is independently
selected from hydrogen or C.sub.1-C.sub.4 alkyl, more preferably
each R.sup.1 is hydrogen.
[0109] Yet a further synthetic method, according to the following
scheme, comprises converting a pyruvate salt or an analog thereof
to the respective pyruvate analog of Formula (IIIb) as follows:
##STR00029##
[0110] Preferably, the solvent is a polar aprotic solvent. More
preferably, the solvent is dimethylformamide.
[0111] In a preferred embodiment, each R.sup.1 is independently
selected from hydrogen or C.sub.1-C.sub.4 alkyl, more preferably
each R.sup.1 is hydrogen.
[0112] Preferably R.sup.2X is R.sup.2Br. More preferably, R.sup.2X
is benzyl bromide.
[0113] This method of converting an isotopically labeled pyruvate
salt to the respective pyruvate analog is particularly useful in
isolating or purifying pyruvate salts from materials which contain
dimerized pyruvic acid.
[0114] The compounds of Formula (IIIb), formed occurring to the
reaction, are separated by techniques known in the art, preferably
by distillation or chromatography.
[0115] The compounds of Formula (IIIa) synthesized by this process
can then be converted to the respective isotopically labeled
pyruvic acid analogs as described previously or even converted to
the same pyruvate salt (but having a higher purity than the
original pyruvate salt).
[0116] Accordingly, one synthetic route to pyruvic acid or the
intermediates of pyruvic acid using the compounds generally
described in Formulas (I), (II), and (III) is as follows:
##STR00030##
[0117] In general, magnesium turnings are combined with Formula IIa
to yield the Gringard reagent Formula IIb. Formula IIb is then
reacted in the presence of isotopically labeled carbon dioxide
(wherein the oxygen atoms may optionally be isotopically labeled)
to yield the intermediate of Formula IIIa'. This reaction can be
run in an organic solvent selected from ether and related solvents
including tetrahydrofuran, dioxane, glymes and the like.
[0118] The reaction is preferably run with cooling and more
preferably at a temperature of about 0.degree. C. or below, more
preferably about -50.degree. C. or below. The reaction is also
preferably run in an inert atmosphere.
[0119] Intermediate Formula IIIa' is then reacted with a weak base
in a solvent including methylene chloride, THF, or acetone at room
temperature or above and with a reagent having a halogenated
leaving group to arrive at the compound of Formula IIIa. One of
ordinary skill in the art would be able to determine the necessary
reagent having a halogenated leaving to arrive at the desired
pyruvate derivative. For example, the reagent having a halogenated
leaving group may be selected from benzyl chloride. Bases useful in
the processes of the present invention include K.sub.2CO.sub.3,
NaHCO.sub.3, Li.sub.2CO.sub.3, Cs.sub.2CO.sub.3, t-ButOK, t-ButOLi,
hydroxides, metal hydrides, and alkyl salts.
[0120] Ozonolysis is then carried out in the presence of Formula
IIIIa to yield the compound of Formula IIIb. Preferably, this
reaction can be run in a C.sub.1-C.sub.6 alcohol (straight chain or
branched), methylene chloride, chloroform and the like at a reduced
temperature, more preferably at about 0.degree. C. or below.
[0121] Finally, the compound of Formula IIIb is hydrogenated to
yield the isotopically labeled pyruvic acid of Formula V. This can
be run in a C.sub.1-C.sub.6 alcohol (straight chain or branched), a
ketone, water, an ether, or mixtures thereof. The reaction is
preferably run under a pressure of about 4 to about 12 psig, more
preferably run using a palladium catalyst, even more preferably on
charcoal. The hydrogenation could also be run in acetone.
[0122] This step could also be undertaken using by means of acid
hydrolysis under conditions known to those of skill in the art.
[0123] Although the scheme shown above is one method of producing
pyruvic acid, one of skill in the art would understand that any of
the intermediates described above can be synthesized, isolated, and
recovered independently of one another. The intermediates may be
used to produce pyruvic acid intermediates or other compounds not
disclosed herein.
[0124] Isotopically labeled pyruvic acid analogs can also be
prepared by hydrolyzing compounds having Formula (IV) under acidic
conditions. By means of example, the production of
[1-.sup.13C]pyruvic acid by means of acid hydrolysis is illustrated
below.
##STR00031##
[0125] Compounds having Formula (IV) can be produced from
isotopically labeled N,N-dialkyl-2-oxo-oxamates by Grignard
addition, as follows:
Synthesis of [1-.sup.13C]Propanamide, N,N-dialkyl-2-Oxo
##STR00032##
[0126] wherein R is as defined as R.sup.2 in Formula (IV).
Synthesis of [2-.sup.13C]Propanamide, N,N-dialkyl-2-Oxo
##STR00033##
[0127] wherein R is as defined as R.sup.2 in Formula (IV).
Synthesis of [1,2-.sup.13C.sub.2]Propanamide, N,N-dialkyl-2-Oxo
##STR00034##
[0128] wherein R is as defined as R.sup.2 in Formula (IV).
[0129] Starting materials for the above conversion to compounds
having Formula (IV) may be produced in accordance with U.S. Pat.
No. 6,753,446, incorporated herein by reference. For example, an
oxamide may be prepared according to the following:
[.sup.13C]Methyl phenyl sulfide was reacted with sec-butyl lithium
followed by [.sup.13C]carbon dioxide to form intermediate (I). This
intermediate (I) was then reacted with oxalyl chloride followed by
dimethyl amine to form intermediate (II). This intermediate (II)
was then reacted with sulfuryl chloride followed by 10 percent
water in ethanol to form [.sup.1-.sup.13C]acetic acid,
(dimethylamino)oxo-, ethyl ester.
[0130] Compounds of Formula (IV) can also be prepared by reacting
compounds having Formula (IIIa') with oxalyl chloride to form an
acid halide intermediate, followed by reaction with an amine to
form the di-substituted amide having Formula (IV). For example, the
reaction with oxalyl chloride, and amine, proceeds as follows:
##STR00035##
[0131] Preferably, the reaction with oxalyl chloride is run in a
non-polar aprotic solvent, more preferably dichloromethane.
Preferably, the reaction with the amine is run in a non-polar
solvent, more preferably THF.
[0132] Synthetic examples of the production of isotopically labeled
benzyl pyruvate and isotopically labeled pyruvic acid are as
follows:
Step 1: Synthesis of [1-.sup.13C]methacrylic acid
##STR00036##
[0134] An oven dried 2 L 3-neck round bottom flask equipped with a
300 mm Allihn reflux condenser with gas adapter, heating mantle,
125 mL addition funnel with septa, and mechanical stirrer was
placed under vacuum and back filled with Argon. Isopropenyl
magnesium bromide (0.6 moles) was then added to the round-bottom
flask, and cooled and maintained at--keep with 78 C with a dry
ice/acetone bath. The .sup.13CO.sub.2 was bubbled into the Grignard
via a needle and measured with a flow meter set at about 200 mL per
minute, 27 g, 0.6 moles. The addition took about 55 minutes. After
addition, the reaction was stirred for fifteen minutes. Meanwhile,
100 mL of concentrated 12M HCl, 1.2 moles, was diluted with 100 mL
of water and transferred to the addition funnel in portions and
added as a steady stream to the Grignard over about ten minutes.
After addition, the cold bath was removed and replaced with water,
to warm the reaction to room temperature. The resulting mostly
colorless biphasic mixture was transferred to a 2 L separatory
funnel. The lower aqueous phase was made acidic by addition of 0.6
moles of HCl. The aqueous was extracted with dichloromethane
(3.times.200 mL) and was separated. The organic layers were
combined and washed with 0.7 moles of NaOH. The aqueous layer was
separated and evaporated to dryness. The [1-.sup.13C]sodium
methacylate was used in the subsequent reaction without
purification.
Step 2: Synthesis of Benzyl [1-.sup.13C]methacrolate
##STR00037##
[0136] The [1-.sup.13C]sodium methacylate was added to dimethyl
formamide (DMF) (250 mL) in a 2 L round bottom flask at room
temperature. This mixture was allowed to stir for about five
minutes and then benzyl chloride (77.195 g, 0.6098 mol, 70.18 mL)
was added at a quick drip rate over a twelve minute period. The
reaction proceeded for six hours. Dichloromethane (750 mL) was
added to the mixture and this mixture was filtered through a frit
funnel. It was then transferred to a separatory funnel and was
washed with DI water (3.times.150 mL). The last wash (4th) was done
using sodium thiosulfate (15 g in 150 mL) to remove iodine from the
solution. The organic extract was dried over sodium sulfate and
evaporated in vacuo to a slightly yellow oil containing the desired
product with some DMF. The DMF was removed under vacuum by heating
the flask to 40oC until about all of the DMF solvent was
removed.
Step 3: Synthesis of Benzyl [1-13C]pyruvate
##STR00038##
[0138] A stirred solution of 25 mL (147.5 mmole) of benzyl
methacrylate in 500 mL of dichloromethane and 125 mL of methanol
was cooled to -78.degree. C. and ozonized until the solution was
pale blue, indicating excess ozone. The solution was purged with
nitrogen until the blue color of ozone had dissipated, and then
14.1 mL (192 mmole, 1.3 equivalents) of dimethyl sulfide was added
rapidly dropwise under nitrogen. After stirring for one hour more
at -78.degree. C. the solution was removed from the cold bath and
allowed to stir at room temperature for 3 hours. The solution may
be stored overnight in the freezer at this point. Volatiles were
removed on the rotary evaporator at 40.degree. C. and the residue
was taken up in 100 mL of dichloromethane and washed with 100 mL of
water to remove dimethyl sulfoxide. The water layer was back
extracted with a small volume of dichloromethane. The combined
organics were washed with water in this fashion twice more. The
final organic layer was filtered through cotton, concentrated on
the rotary evaporator, and high-vacuum dried leaving an essentially
quantitative yield of benzyl pyruvate as a colorless liquid. A
small amount of formaldehyde methyl hemiacetal and/or the methyl
hemiacetal of benzyl pyruvate may be present but do not interfere
in the next step.
Step 4: Synthesis of [1-13C]Pyruvic Acid
##STR00039##
[0140] 5 g (28 mmole) of the above benzyl pyruvate was dissolved in
100 mL of absolute ethanol in a heavy-walled bottle and blanketed
with nitrogen. 0.5 g of 51 palladium on charcoal catalyst was added
and the mixture was in a Parr shaker hydrogenation apparatus. The
mixture was deaerated three times by evacuation followed by
refilling with hydrogen. Hydrogenation was then commenced at 8 psi
for one hour. Hydrogen was then removed by evacuation followed by
refilling with nitrogen. Catalyst was removed by vacuum filtration
through a bed of Celite. The filter cake was washed with ethanol
and the colorless to pale yellow filtrate was concentrated on the
rotary evaporator at room temperature. Excessive vacuum or higher
temperature must be avoided to prevent loss of product. The product
obtained still contained a little ethanol and showed pyruvic acid,
varying amounts of its ethyl hemiacetal, and a very small amount of
ethyl pyruvate by NMR. Water may be added and distilled off at a
bath temperature of less than 50.degree. C. under high vacuum with
liquid nitrogen cooling of the receiver. After most of the water
was removed, additional water was added to the pot and the process
repeated. What remained was a concentrated solution of pyruvic acid
and its hydrate possibly contaminated by a little formaldehyde
hydrate and ethyl pyruvate. Titration with 1N aqueous sodium
hydroxide to an endpoint of pH 5.8 and removal of water and other
volatiles yielded solid sodium pyruvate.
[0141] The reaction above could also be run in other solvents for
example the reaction was run using acetone and at the end of the
process [1-13C]Pyruvic acid was isolated as a mixture of 2 parts
pyruvic acid to 1 of acetone. For example, the Hydrogenation of
benzyl [1-.sup.13C]pyruvate using acetone is accomplished as
follows: Benzyl [1-.sup.13C]pyruvate (5.1 g, 0.0285 moles) was
dissolved in acetone (51 mL) and placed in a hydrogenation vessel
which had 10% Pd/carbon (0.28 g). The reaction was purged with
argon and then the reaction was evacuated under vacuum. The
reaction vessel was filled with hydrogen to 10 psi and then shaken
for 24 hours. The reaction was filtered to remove the catalyst and
then concentrated. This mixture, which now contained toluene and
[1-.sup.13C]pyruvic acid, was treated with hexane. The layers were
then separated and the hexane layer which contained the
[1-.sup.13C]pyruvic acid was evaporated to give a quantitative
yield of the desired product as a mixture of acetone to
[1-.sup.13C]pyruvic acid (1:2).
[0142] Shelf Stability of Isotopically Labeled Pyruvic Acid:
[0143] Samples of isotopically labeled [1-.sup.13C]pyruvic acid,
having Formula VI and synthesized as discussed immediately below,
were subjected to various conditions as depicted in Table 1, in
order to establish the compound's shelf storage stability.
##STR00040##
[0144] The [1-.sup.13C]pyruvate of Formula VI was synthesized as
follows:
[0145] Benzyl [1-.sup.13C]pyruvate, Formula VIA, was synthesized
according to the methods discussed previously. The benzyl
[1-.sup.13C]pyruvate was hydrogenated and the hydrogenation mixture
was diluted with water and then extracted with dichloromethane. The
organic phase contained toluene and some methanol while the aqueous
phase contained the [1-.sup.13C]pyruvic acid and methanol. The
aqueous phase was then distilled until all of the methanol was
removed, thereby leaving [1-.sup.13C]pyruvic acid (purity: 98+/-2%,
by NMR).
##STR00041##
[0146] The resulting [1-.sup.13C]pyruvic acid was tested as follows
in Table 1:
TABLE-US-00001 TABLE 1 Samples of isotopically labeled pyruvic acid
were subjected to various storage conditions. Condition Duration
Sample Temperature Storage 12 weeks 17 weeks 1 -10.degree. C. Dark
Stable Stable 2 4.degree. C. Dark Stable Stable 3 Room Dark Stable
Stable temperature* 4 Room Light Stable Stable temperature* *Room
temperature denotes a temperature ranging from about 22.degree. C.
to about 26.degree. C.
[0147] .sup.1H and .sup.13C NMR spectra of the distilled
[1-.sup.13C]pyruvic acid in water (0.025M) were acquired at the
onset of the study and prior to exposing the samples to the various
conditions specified in Table 1. .sup.1H and .sup.13C NMR spectra
were again acquired for each of the samples after being exposed to
the aforementioned conditions on a weekly basis for 12-weeks. The
spectra acquired after exposure were compared visually and appeared
to remain unchanged as compared with the spectra acquired prior to
exposure, i.e. the spectra were as expected for [1-.sup.13C]pyruvic
acid in water. As such, it was concluded that each of the samples
were shelf stable. Similarly, .sup.1H and .sup.13C NMR spectra
acquired after 17-weeks of exposure remained unchanged as compared
with the spectra acquired prior to exposure. Once again, it was
concluded that each of the samples were shelf stable under the
conditions provided in Table 1.
[0148] Concentration studies were also performed to show that
[1-.sup.13C]pyruvic acid solutions would also be expected to be
stable at higher concentrations. To demonstrate such stability, the
[1-.sup.13C]pyruvic acid solutions were compared to commercial
samples of pyruvic acid (Formula VIB) obtained from Sigma-Aldrich
Co. in water at higher concentrations. Commercial pyruvic acid was
dissolved in water to arrive at the various concentrations listed
in Table 2 below. Each of these commercial solutions were subjected
to light for 6-weeks at temperatures ranging from about 22.degree.
C. to about 26.degree. C. At each of the concentrations listed
below, it was discovered that the pyruvic acid solutions were
stable. As the purity of the commercial pyruvic acid samples were
comparable to the purity of the materials produced in accordance
with the present invention, and as the commercial materials were
stable in water for at least 6 weeks at relatively high
concentrations, it is believed that [1-.sup.13C]pyruvic acid
solutions having at least those concentrations in water would also
be stable.
TABLE-US-00002 TABLE 2 Pyruvic acid, of varying concentrations, was
found to be stable after 6-weeks. (Formula VIB) ##STR00042##
Concentration After 6-weeks 11.3 Stable 5.7 Stable 3.8 Stable 2.8
Stable 2.3 Stable
[0149] The molarity of sample 4 was confirmed to be 0.025M by
titrating the aqueous sample with sodium hydroxide. The titration
with the sodium hydroxide also revealed that in acidic conditions,
such as at pHs ranging from about 5.8 to about 1, pyruvic acid may
be present either as the acid or as sodium [1-.sup.13C]pyruvate,
depending on the sodium hydroxide concentration. It was also
discovered that at pHs greater than 5.8, such as at a pH of about
6.2, some of the pyruvic acid was present in the form of a
dimerized sodium salt. For example, at a pH of about 6.2, the pH
attained in titrating the solution of sample 4, HPLC revealed that
sample 4 consisted of 89% sodium [1-.sup.13C]pyruvate and about
2-4% dimerized [1-.sup.13C]pyruvate salt (accounting for about 4-6%
of the pyruvic acid). As such, it was discovered that to minimize
dimmer formation, it may be necessary to control the pH of
[1-.sup.13C]pyruvate solutions, and in particular, any aqueous
solution should be maintained at a pH of about 6.5 or less, more
preferably 6.0 or less, and even more preferably 5.8 or less.
[0150] Although the invention herein has been described with
reference to particular embodiments, it is to be understood that
these embodiments are merely illustrative of the principles and
applications of the present invention. It is therefore to be
understood that numerous modifications may be made to the
illustrative embodiments and that other arrangements may be devised
without departing from the spirit and scope of the present
invention as defined by the appended claims.
* * * * *