U.S. patent application number 12/040470 was filed with the patent office on 2009-07-09 for isotopically enriched n-substituted piperazine acetic acids and methods for the preparation thereof.
Invention is credited to James M. Coull, Subhakar Dey, Darryl J.C. Pappin, Sasi Pillai, Subhasish Purkayastha.
Application Number | 20090176984 12/040470 |
Document ID | / |
Family ID | 34711414 |
Filed Date | 2009-07-09 |
United States Patent
Application |
20090176984 |
Kind Code |
A1 |
Dey; Subhakar ; et
al. |
July 9, 2009 |
Isotopically Enriched N-Substituted Piperazine Acetic Acids And
Methods For The Preparation Thereof
Abstract
In some embodiments, this invention pertains to isotopically
enriched N-substituted piperazine acetic acids. In some
embodiments, this invention pertains to methods for the preparation
of isotopically enriched N-substituted piperazine acetic acids.
Inventors: |
Dey; Subhakar; (Billerica,
MA) ; Pappin; Darryl J.C.; (Boxborough, MA) ;
Purkayastha; Subhasish; (Acton, MA) ; Pillai;
Sasi; (Littleton, MA) ; Coull; James M.;
(Westford, MA) |
Correspondence
Address: |
Applied Biosystems;Mila Kasan
850 Lincoln Center Drive
Foster City
CA
94404
US
|
Family ID: |
34711414 |
Appl. No.: |
12/040470 |
Filed: |
February 29, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10751387 |
Jan 5, 2004 |
7355045 |
|
|
12040470 |
|
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|
Current U.S.
Class: |
544/399 |
Current CPC
Class: |
C07D 403/12 20130101;
C07D 295/03 20130101; C07D 295/15 20130101 |
Class at
Publication: |
544/399 |
International
Class: |
C07D 241/04 20060101
C07D241/04 |
Claims
1. An isotopically enriched N-substituted piperazine acetic acid
compound of the formula: ##STR00031## , or a salt thereof, wherein;
X is O or S; Y is a straight chain or branched C1-C6 alkyl group or
a straight chain or branched C1-C6 alkyl ether group wherein the
carbon atoms of the alkyl group or alkyl ether group each
independently are optionally substituted with linked deuterium or
fluorine atoms; each Z is independently hydrogen, deuterium,
fluorine, chlorine, bromine, iodine, an amino acid side chain, a
straight chain or branched C1-C6 alkyl group that may optionally
contain a substituted or unsubstituted aryl group wherein the
carbon atoms of the alkyl and aryl groups each independently are
optionally substituted with linked deuterium or fluorine atoms, a
straight chain or branched C1-C6 alkyl ether group that may
optionally contain a substituted or unsubstituted aryl group
wherein the carbon atoms of the alkyl and aryl groups each
independently are optionally substituted with linked deuterium or
fluorine atoms or a straight chain or branched C1-C6 alkoxy group
that may optionally contain a substituted or unsubstituted aryl
group wherein the carbon atoms of the alkoxy and aryl groups each
independently are optionally substituted with linked deuterium or
fluorine atoms.
2. The compound of claim 1, wherein the N-substituted piperazine
acetic acid is isotopically enriched with two or more heavy atom
isotopes.
3. The compound of claim 1, wherein the N-substituted piperazine
acetic acid is isotopically enriched with three or more heavy atom
isotopes.
4. The compound of claim 1, wherein the N-substituted piperazine
acetic acid is isotopically enriched with four or more heavy atom
isotopes.
5. The compound of claim 2, wherein the heavy atom isotopes are
each independently .sup.18O, .sup.15N or .sup.13C, but not
deuterium.
6. The compound of claim 1, wherein each Z is independently
hydrogen, fluorine, chlorine, bromine or iodine.
7. The compound of claim 1, wherein each Z is independently
hydrogen, methyl or methoxy.
8. The compound of claim 1, wherein Y is methyl, ethyl, n-propyl,
isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl.
9. The compound of claim 1, wherein X is .sup.16O or .sup.18O.
10. The compound of claim 1, wherein each nitrogen atom of the
piperazine ring is independently .sup.14N or .sup.15N.
11. The compound of claim 1 of the formula: ##STR00032## wherein
each C* is independently .sup.12C or .sup.13C; X is O or S; Y is a
straight chain or branched C1-C6 alkyl group or a straight chain or
branched C1-C6 alkyl ether group wherein the carbon atoms of the
alkyl group or alkyl ether group each independently are optionally
substituted with linked deuterium or fluorine atoms; each Z is
independently hydrogen, deuterium, fluorine, chlorine, bromine,
iodine, an amino acid side chain, a straight chain or branched
C1-C6 alkyl group that may optionally contain a substituted or
unsubstituted aryl group wherein the carbon atoms of the alkyl and
aryl groups each independently are optionally substituted with
linked deuterium or fluorine atoms, a straight chain or branched
C1-C6 alkyl ether group that may optionally contain a substituted
or unsubstituted aryl group wherein the carbon atoms of the alkyl
and aryl groups each independently are optionally substituted with
linked hydrogen, deuterium or fluorine atoms or a straight chain or
branched C1-C6 alkoxy group that may optionally contain a
substituted or unsubstituted aryl group wherein the carbon atoms of
the alkyl and aryl groups each independently are optionally
substituted with linked hydrogen, deuterium or fluorine atoms.
12. The compound of claim 1 of the formula: ##STR00033## or a salt
thereof.
13. The compound of claim 12, wherein the compound is a zwitterion,
mono-TFA salt, a mono-HCl salt, a bis-TFA salt or a bis-HCl
salt.
14. The compound of claim 12, wherein each incorporated heavy atom
isotope is present in at least 80 percent isotopic purity.
15. The compound of claim 12, wherein each incorporated heavy atom
isotope is present in at least 93 percent isotopic purity.
16. The compound of claim 12, wherein each incorporated heavy atom
isotope is present in at least 96 percent isotopic purity.
17. The compound of claim 1, wherein the N-substituted piperazine
acetic acid is a mono-TFA salt, a mono-HCl salt, a bis-HCl salt or
a bis-TFA salt.
18. The compound of claim 1, wherein each incorporated heavy atom
isotope is present in at least 80 percent isotopic purity.
19. The compound of claim 1, wherein each incorporated heavy atom
isotope is present in at least 93 percent isotopic purity.
20. The compound of claim 1, wherein each incorporated heavy atom
isotope is present in at least 96 percent isotopic purity.
21. The compound of claim 12, wherein the compound is a carboxylate
anion.
22. The compound of claim 1, wherein the compound is a carboxylate
anion.
23. An isotopically enriched N-substituted piperazine acetic acid
compound of the formula: ##STR00034## or a salt thereof, wherein
each X is O or S; Y is a straight chain or branched C1-C6 alkyl
group or a straight chain or branched C1-C6 alkyl ether group
wherein the carbon atoms of the alkyl group or alkyl ether group
each independently are optionally substituted with linked deuterium
or fluorine atoms; and each Z is independently hydrogen, fluorine,
chlorine, bromine, iodine, an amino acid side chain or a straight
chain or branched C1-C6 alkyl group that may optionally contain a
substituted or unsubstituted aryl group wherein the carbon atom of
the alkyl and aryl groups each independently are optionally
substituted with linked fluorine atoms; wherein the N-substituted
piperazine acetic acid is isotopically enriched with one or more
.sup.13C atoms and/or .sup.15N atoms.
24. The compound of claim 23, wherein Y is a straight chain or
branched C1-C6 alkyl group and each Z is independently hydrogen,
fluorine, chlorine, bromine, iodine, an amino acid side chain or
straight chain or branched C1-C6 alkyl group.
25. An isotopically enriched N-substituted piperazine acetic acid
compound of the formula: ##STR00035## or a salt thereof, wherein; X
is O or S; Y is a straight chain or branched C1-C6 alkyl group or a
straight chain or branched C1-C6 alkyl ether group; and each Z is
independently hydrogen, fluorine, chlorine, bromine, iodine, an
amino acid side chain or a straight chain or branched C1-C6 alkyl
group; and wherein the N-substituted piperazine is isotopically
enriched with one or more .sup.13C atoms, .sup.15N atoms and/or
.sup.18O atoms.
26. An isotopically enriched N-substituted piperazine acetic acid
compound of the formula: ##STR00036## or a salt thereof, wherein
the N-substituted piperazine is isotopically enriched with one or
more .sup.13C atoms, .sup.15N atoms and/or .sup.18O atoms.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of co-pending application
Ser. No. 10/751,387 filed Jan. 5, 2004 and incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] In some embodiments, this invention pertains to isotopically
enriched N-substituted piperazine acetic acids. In some
embodiments, this invention pertains to methods for the preparation
of isotopically enriched N-substituted piperazine acetic acids.
INTRODUCTION
[0003] In some embodiments, this invention pertains to isotopically
enriched N-substituted piperazine acetic acids. In some
embodiments, this invention pertains to methods for the preparation
of isotopically enriched N-substituted piperazine acetic acids.
N-substituted piperazine acetic acids can be intermediates in the
preparation of active esters of N-substituted piperazine acetic
acid. Active esters are well known in peptide synthesis and refer
to certain esters that are easily reacted with an amine of an amino
acid under conditions commonly used in peptide synthesis (For a
discussion of active esters please see: Innovation And Perspectives
In Solid Phase Synthesis, Editor: Roger Epton, SPCC (UK) Ltd,
Birmingham, 1990).
[0004] The active esters of N-substituted piperazine acetic acid
can be used as labeling reagents. In some embodiments, a set of
isobaric labeling reagents can be prepared. The set of isobaric
labeling reagents can be used to label analytes, such as peptides,
proteins, amino acids, oligonucleotides, DNA, RNA, lipids,
carbohydrates, steroids, small molecules and the like. The labeled
analytes can be mixed together and analyzed simultaneously in a
mass spectrometer. Because the heavy atom isotope distribution in
each of the isobaric labeling reagents can be designed to result in
the generation of a unique "signature ion" when analyzed in a mass
spectrometer (MS), labeled components of the mixture associated
with each of the labeling reagents, and by implication components
of each labeling reaction used to produce the mixture, can be
deconvoluted. Deconvolution can include determining the relative
and/or absolute amount of one or more labeled components in each of
the individual samples that were labeled and combined to form the
mixture. The N-substituted piperazine acetic acid active esters
described herein therefore can be powerful tools for analyte
analysis, including but not limited to multiplex proteomic
analysis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0005] FIG. 1 is an illustration of a synthetic scheme for the
synthesis of N-methyl piperazines.
[0006] FIG. 2A is an illustration of a synthetic scheme for the
synthesis of N-methyl piperazine acetic acids.
[0007] FIG. 2B is an illustration of another synthetic scheme for
the synthesis of N-methyl piperazine acetic acids.
[0008] FIG. 2C is an illustration of yet another synthetic scheme
for the synthesis of N-methyl piperazine acetic acids.
[0009] FIG. 3A is an illustration of a synthetic scheme for the
synthesis of .sup.18O labeled N-methyl piperazine acetic acids.
[0010] FIG. 3B is an illustration of another synthetic scheme for
the synthesis of .sup.18O labeled N-methyl piperazine acetic
acids.
[0011] FIG. 4A is an illustration of a synthetic scheme for the
synthesis of various active esters of N-methyl piperazine acetic
acid.
[0012] FIG. 4B is an illustration of another synthetic scheme for
the synthesis of various active esters of N-methyl piperazine
acetic acid.
[0013] FIG. 4C is an illustration of yet another synthetic scheme
for the synthesis of various active esters of N-methyl piperazine
acetic acid.
[0014] FIG. 4D is an illustration of still another synthetic scheme
for the synthesis of various active esters of N-methyl piperazine
acetic acid.
[0015] FIG. 5A is an illustration of the heavy atom isotope
incorporation pathway for the preparation of four isobaric N-methyl
piperazine acetic acids.
[0016] FIG. 5B is an illustration of the labeling and fragmentation
of peptides using four isobaric N-methyl piperazine acetic acid
active ester labeling reagents.
DEFINITIONS
[0017] For the purposes of interpreting of this specification, the
following definitions will apply and whenever appropriate, terms
used in the singular will also include the plural and vice
versa:
[0018] As used herein, "analyte" refers to a molecule of interest
that may be determined. Non-limiting examples of analytes include,
but are not limited to, proteins, peptides, nucleic acids (both DNA
or RNA), carbohydrates, lipids, steroids and other small molecules
with a molecular weight of less than 1500 Daltons (Da). The source
of the analyte, or the sample comprising the analyte, is not a
limitation as it can come from any source. The analyte or analytes
can be natural or synthetic. Non-limiting examples of sources for
the analyte, or the sample comprising the analyte, include cells or
tissues, or cultures (or subcultures) thereof. Non-limiting
examples of analyte sources include, but are not limited to, crude
or processed cell lysates, body fluids, tissue extracts, cell
extracts or fractions (or portions) from a separations process such
as a chromatographic separation, a 1D electrophoretic separation, a
2D electrophoretic separation or a capillary electrophoretic
separation. Body fluids include, but are not limited to, blood,
urine, feces, spinal fluid, cerebral fluid, amniotic fluid, lymph
fluid or a fluid from a glandular secretion. By processed cell
lysate we mean that the cell lysate is treated, in addition to the
treatments needed to lyse the cell, to thereby perform additional
processing of the collected material. For example, the sample can
be a cell lysate comprising one or more analytes that are peptides
formed by treatment of the cell lysate with a proteolytic enzyme to
thereby digest precursor peptides and/or proteins.
[0019] Except as when clearly not intended based upon the context
in which it is being used (e.g. when made in reference to a
structure that dictates otherwise), "ester" refers to both an ester
and/or a thioester.
[0020] As used herein, "fragmentation" refers to the breaking of a
covalent bond.
[0021] As used herein, "fragment" refers to a product of
fragmentation (noun) or the operation of causing fragmentation
(verb).
[0022] As used herein, "isotopically enriched" means that a
compound (e.g. labeling reagent) has been enriched synthetically
with one or more heavy atom isotopes (e.g. stable isotopes such as
Deuterium, .sup.13C, .sup.15N, .sup.18O, .sup.37Cl or .sup.81Br).
Because isotopic enrichment is not 100% effective, there can be
impurities of the compound that are of lesser states of enrichment
and these will have a lower mass. Likewise, because of
over-enrichment (undesired enrichment) and because of natural
isotopic abundance, there can be impurities of greater mass.
[0023] As used herein, "labeling reagent" refers to a moiety
suitable to mark an analyte for determination. The term label is
synonymous with the terms tag and mark and other equivalent terms
and phrases. For example, a labeled analyte can be referred to as a
tagged analyte or a marked analyte.
[0024] As used herein, "natural isotopic abundance" refers to the
level (or distribution) of one or more isotopes found in a compound
based upon the natural prevalence of an isotope or isotopes in
nature. For example, a natural compound obtained from living plant
matter will typically contain about 0.6% .sup.13C.
DESCRIPTION OF VARIOUS EMBODIMENTS OF THE INVENTION
I. Preparation of N-Substituted Piperazines Comprising Heavy Atom
Isotopes
[0025] In some embodiments, this invention pertains to a method for
the production of isotopically enriched N-substituted piperazines,
and the N-substituted piperazines themselves. According to the
method, a partially protected amino acid can be condensed with an
N-substituted amino acid ester wherein at least one of the two
amino acids comprises a heavy atom isotope such as, for example,
.sup.18O, .sup.15N, .sup.13C, .sup.81Br, .sup.37Cl or deuterium.
When condensing the two amino acids, any side chain reactive groups
can be protected as they would be for the condensation of amino
acids to form peptides. Similarly, the condensation chemistry can
be chosen from the various methods known for condensing amino
acids. These include, but are not limited to, the use of
carbodiimides (e.g. dicyclohexylcarbodiimide, DCC), active esters,
mixed anhydride formation and the like.
[0026] The partially protected amino acid comprises an
amine-protecting group (N-protecting group), such as
tert-butyloxycarbonyl (t-boc); a well-known protecting group in
peptide synthesis. The partially protect amino acid can comprise a
side chain protecting where the amino acid comprises a reactive
side chain moiety. The amino acid can be any natural amino acid
(e.g. glycine, alanine, lysine) or non-natural amino acid of basic
structure:
##STR00001##
wherein Pg can be the N-protecting group. Each group Z can be
independently hydrogen, deuterium, fluorine, chlorine, bromine,
iodine, an amino acid side chain, a straight chain or branched
C1-C6 alkyl group that may optionally contain a substituted or
unsubstituted aryl group wherein the carbon atoms of the alkyl and
aryl groups each independently comprise linked hydrogen, deuterium
or fluorine atoms, a straight chain or branched C1-C6 alkyl ether
group that may optionally contain a substituted or unsubstituted
aryl group wherein the carbon atoms of the alkyl and aryl groups
each independently comprise linked hydrogen, deuterium or fluorine
atoms or a straight chain or branched C1-C6 alkoxy group that may
optionally contain a substituted or unsubstituted aryl group
wherein the carbon atoms of the alkyl and aryl groups each
independently comprise linked hydrogen, deuterium or fluorine
atoms. In some embodiments, each Z is independently hydrogen,
methyl or methoxy. In some embodiments, each Z is hydrogen,
deuterium, fluorine, chlorine, bromine or iodine. An alkyl ether
group, as used herein, can include one or more polyethylene glycol
substituents. Similarly, the alkoxy group, as used herein, can
comprise ether and/or polyethylene glycol substituents. The
N-protecting group can be an acid labile protecting group. The
N-protecting group can be a base labile protecting group.
[0027] The N-substituted amino acid ester can be any natural amino
acid (e.g. glycine, alanine, lysine) or non-natural amino acid of
basic structure:
##STR00002##
wherein Z is previously defined above. The group Y can be a
straight chain or branched C1-C6 alkyl group or a straight chain or
branched C1-C6 alkyl ether group wherein the carbon atoms of the
alkyl group or alkyl ether group each independently comprise linked
hydrogen, deuterium or fluorine atoms. In some embodiments, Y is
methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or
tert-butyl. The group R can be a straight chain or branched C1-C6
alkyl group or a substituted or unsubstituted phenyl group, wherein
the carbon atoms of the alkyl group or phenyl group each
independently comprise linked hydrogen, deuterium or fluorine
atoms. In some embodiments, the N-substituted amino acid ester is
the ester (e.g. methyl or ethyl) of sarcosine, which is an ester of
N-methyl glycine.
[0028] Every possible permutation of .sup.15N or .sup.13C labeled
glycine is commercially available. Likewise, other natural amino
acids are commercially available with one or more incorporated
heavy atom isotopes. Because glycine, and other amino acids,
comprising one or more heavy atom isotopes are commercially
available, these amino acids can be easily incorporated into the
procedure for the production of N-substituted piperazines. The
amino acids comprising heavy atom isotopes can be N-protected using
procedures well-known in peptide chemistry. For example, the amino
acids can be N-protected with a 9-fluorenylmethoxycarbonyl (Fmoc)
group or a t-boc group. Furthermore the amino acids comprising
heavy atom isotopes can be N-alkylated and converted to an ester of
the amino acid using well-known procedures. Accordingly, heavy atom
isotope containing starting materials for the preparation of
N-substituted piperazines, as described herein, are either
commercially available, or can be easily prepared from commercially
available amino acids using no more than routine
experimentation.
[0029] According to the method, the two amino acids can be
condensed to thereby produce an N-protected peptide dimer as an
ester. The N-protected peptide dimer ester can comprise one or more
heavy atom isotopes via the incorporation of the one or more amino
acids comprising one or more heavy atom isotopes. The N-protected
peptide dimer ester can comprise one heavy atom isotope, two heavy
atom isotopes, three heavy atom isotopes, four heavy atom isotopes,
five heavy atom isotopes or six heavy atom isotopes. The
N-protected peptide dimer ester can have the general formula:
##STR00003##
wherein Pg, R, Y and Z are previously defined.
[0030] According to the method, the N-protected peptide dimer ester
can then be cyclized to form a 6-membered cyclic dione. Cyclization
proceeds by removing the N-protecting group of the N-protected
peptide dimer ester and driving the reaction of the deprotected
amine with the ester group. The reaction can be carried out under
basic conditions and can be heated to speed production of the
product. The product of the cyclization can have the general
formula:
##STR00004##
wherein Y and Z are previously defined.
[0031] According to the method, the ketone groups of the cyclic
dione can then be reduced to form the desired N-substituted
piperazine comprising one or more heavy atom isotopes. The
reduction can be performed using a reducing agent, such as lithium
aluminum hydride (LAH) or Red-Al (Sigma-Aldrich). The product, in
some embodiments being a volatile oil, can be optionally
temporarily modified (e.g. protected) to aid in isolation. Because
piperazine comprises two basic nitrogen atoms, the product can, in
some embodiments, be isolated as a mono or bis-acid salt. For
example, the N-substituted piperazine comprising one or more heavy
atom isotopes can be isolated as a mono-TFA salt, a mono-HCl salt,
a bis-TFA salt or a bis-HCl salt.
[0032] FIG. 1 illustrates the application of the aforementioned
general procedure to the production of N-methyl piperazine.
Examples 1-4 describe the application of the illustrated procedure
to the production of three different N-methyl piperazines each
comprising 1-3 heavy atom isotopes.
[0033] With reference to FIG. 1 and Examples 1-4, t-boc protected
glycine (1) is condensed with sarcosine methyl ester (2) to thereby
produce the dipeptide (3). The t-boc group is removed and the
dipeptide is cyclized to the cyclic dione (4). The ketone groups of
the dione are then reduced to produce N-methyl piperazine. The
N-methyl piperazine product can either be transiently protected (5)
or can be obtained directly from the reduction (6). The product can
also be obtained as a salt (e.g. TFA salt (7) or HCl (8)) of an
acid.
[0034] In summary, a wide variety of N-substituted piperazine
compounds, unlabeled or labeled with one or more heavy atom
isotopes, can be produced by the aforementioned process.
Consequently, the present invention contemplates all possible
isotopically enriched N-substituted piperazine compound comprising
one or more heavy atom isotopes of the general formula:
##STR00005##
including all possible salt forms thereof, wherein Y and Z are
previously defined.
II. Preparation of N-Substituted Piperazine Acetic Acids Comprising
Heavy Atom Isotopes
[0035] In some embodiments, this invention pertains to methods for
the production of isotopically enriched N-substituted piperazine
acetic as well as the isotopically enriched N-substituted
piperazine acetic acids. In some embodiments, an N-substituted
piperazine can be reacted with a halo acetic acid moiety comprising
one or more heavy atom isotopes. In this context, halo refers to
the halogens, chlorine, bromine and iodine. In still some other
embodiments, an N-substituted piperazine comprising one or more
heavy atom isotopes can be reacted with a halo acetic acid moiety.
In some other embodiments, an N-substituted piperazine comprising
one or more heavy atom isotopes can be reacted with a halo acetic
acid moiety comprising one or more heavy atom isotopes.
Accordingly, the heavy atom isotopes found in the product
N-substituted piperazine acetic acids can be introduced by way of
the piperazine, by way of the halo acetic acid moiety or by way of
both the piperazine and the halo acetic acid moiety. As will be
discussed in more detail below, .sup.18O can also be introduced
into the carboxylic acid moiety of an N-substituted piperazine
acetic acid by way of exchange with H.sub.2.sup.18O.
[0036] Numerous light (by light we mean that the compound is not
isotopically enriched with one or more heavy atom isotopes)
N-substituted piperazines (e.g. N-methyl and N-ethyl piperazine)
are commercially available. Furthermore, Section I above describes
the preparation of N-substituted piperazine comprising one or more
heavy atoms from commercially available amino acids. Both light and
heavy (by heavy we mean that the compound has been isotopically
enriched with one or more heavy atom isotopes) N-substituted
piperazine can be used to produce the N-substituted piperazine
acetic acids comprising one or more heavy atom isotopes.
[0037] Numerous light and heavy halo acetic acid moieties are
commercially available. The halo acetic acid moiety to be reacted
with the N-substituted piperazine can be purchased as the
carboxylic acid or as an ester of the carboxylic acid (e.g. the
methyl ester, ethyl ester or phenyl ester). If only the carboxylic
acid is available and the ester is desired, the ester can be
prepared using well-known esterification methods. If only the ester
is available and the carboxylic acid is desired, the ester can be
hydrolyzed to produce the carboxylic acid. Either the carboxylic
acid or the ester can be used in the alkylation reaction provided
that an additional equivalent of base is required if the carboxylic
acid is used. If the ester is used to perform the alkylation, the
product ester can be hydrolyzed to produce the N-substituted
piperazine acetic acid. General structures for the carboxylic acid
and the ester compounds that can be used to alkylate N-substituted
piperazines are:
##STR00006##
wherein Z and R are defined above. Hal is a halogen (Cl, Br or I)
and X is oxygen (O) or sulfur (S). In some embodiments, X is
.sup.16O or .sup.18O. One or more of the atoms of the halo acetic
acid compound can be a heavy atom isotope.
[0038] The alkylation of an N-substituted piperazine with a halo
acetic acid moiety proceeds under basic conditions. The base need
only be strong enough to deprotonate piperazine but can be selected
to not substantially react with the halo acetic acid moiety. In
some embodiments, two or more equivalents of N-substituted
piperazine can be used, as N-substituted piperazine is a base. If
it is desirable to use only one equivalent of N-substituted
piperazine (for example, when the N-substituted piperazine is
labeled with one or more heavy atom isotopes and is therefore
valuable), other bases can be used. Suitable bases include, but are
not limited to, hindered bases such as triethylamine (Et.sub.3N)
and diisopropylethyamine (DIEPA). Other suitable bases in sodium
carbonate and potassium carbonate. Hindered bases are a good choice
because they do not react substantially with the halo acetic acid
moiety.
[0039] A solid phase base, such as
1,5,7-Triazabicyclo[4.4.0]dec-5-ene (TBD) bound to polystyrene
crosslinked with 2% DVB, Capacity (base): .about.2.6 mmol/g
(ss-TBD, Fluka, P/N 90603) can also be used (See FIG. 2B). A solid
phase base has the advantage that it is easily, and completely,
removed from the product by filtration once the alkylation reaction
has been completed. Accordingly, the resulting product is not
contaminated with salt of the base.
[0040] If the carboxylic acid is used to alkylate the N-substituted
piperazine, the reaction can produce a product of the general
formula:
##STR00007##
or a salt thereof, wherein X, Y and Z have been previously defined.
One or more atoms of the N-substituted piperazine acetic acid can
be a heavy atom isotope.
[0041] If the ester is used to alkylate the N-substituted
piperazine, the reaction will produce an ester of the general
formula:
##STR00008##
or a salt thereof, wherein R, X, Y and Z have been previously
defined. One or more atoms of the N-substituted piperazine acetic
acid ester can be a heavy atom isotope. The N-substituted
piperazine acetic acid ester can be converted to the N-substituted
piperazine acetic acid of general formula:
##STR00009##
by hydrolysis of the ester. Depending on the state of protonation
of the ester, it may or may not be necessary to and base to aqueous
solution to perform the hydrolysis because piperazine is basic
(unless neutralized by acid). Accordingly, base can be added as
required to induce the hydrolysis of the ester to the carboxylic
acid, but in some embodiments it will not be required. Hydrolysis
can also be performed under aqueous acidic conditions.
[0042] N-substituted piperazine acetic acid is zwitterionic.
Because it comprises a carboxylic acid group (or thio acid group)
and two basic nitrogen atoms, it can exist in at least four
different forms. It can exist completely deprotonated as its
carboxylate anion. It can exist as its mono protonated zwitterion.
It can exist as a monobasic salt (e.g. mono-TFA or mono-HCl salt).
It can also exist as its dibasic salt (e.g. bis-TFA or bis-HCl
salt). The state of protonation of the product is a function of the
conditions under which it was isolated. All protonation states of
N-substituted piperazine acetic acid are contemplated as
embodiments of the present invention.
[0043] With reference to FIGS. 2A and 2B, as well as Examples 5 and
6, respectively, the production of two different isotopically
enriched N-methyl piperazine acetic acid compounds is described. In
FIG. 2A and Example 5, two equivalents of commercially available
unlabeled N-methyl piperazine is reacted with ethyl bromoacetate to
produce a N-methyl piperazine acetic acid compound comprising two
.sup.13C atoms. Because N-methyl piperazine is basic, hydrolysis of
the ethyl ester proceeded by merely heating the compound in an
aqueous solution.
[0044] With reference to FIG. 2B and Example 6, the starting
piperazine is a bis-TFA salt of .sup.15N labeled N-methyl
piperazine. Acid salts of the piperazine base can be alkylated so
long as sufficient base is added to the reaction to deprotonate
piperazine. In this example, the ethyl bromoacetate is .sup.13C
labeled. Because both the piperazine and acetic acid reactants
comprise heavy atom isotopes, a solid phase base was chosen so that
only one equivalent of each reactant was required to produce the
product. As was observed with Example 5, hydrolysis of the ethyl
ester proceeded by mere heating the compound in an aqueous
solution.
[0045] In some other embodiments, the N-substituted piperazine
acetic acid can be assembled on a solid support. According to the
method and with reference to FIG. 2C, the halo acetic acid moiety,
as a carboxylic acid, can be reached with trityl chloride resin to
thereby produce a support bound halo acetic acid. The support bound
halo acetic acid can then be treated with the desired N-substituted
piperazine (e.g. N-methyl piperazine) under basic conditions to
thereby produce the N-substituted piperazine acetic acid.
Isotopically enriched N-methyl piperazine and halo acetic acid
moieties can be used, including .sup.18O labeled compounds although
.sup.18O labeling can involve special considerations and is
discussed in more detail below.
[0046] In accordance with the aforementioned discussion, a heavy
atom isotope can be incorporated at virtually any position of the
N-substituted piperazine acetic acid, including .sup.18O
incorporation that will be discussed in more detail below.
Consequently, the present invention contemplates all possible
isotopically enriched N-substituted piperazine acetic acids
comprising one or more heavy atom isotopes of the general
formula:
##STR00010##
including all possible salt forms thereof.
III. Incorporation of .sup.18O into N-Substituted Piperazine Acetic
Acids
[0047] In some embodiments, this invention pertains to methods for
the incorporation of .sup.18O into N-substituted piperazine acetic
acids as well as to the .sup.18O labeled N-substituted piperazine
acetic acids themselves. In some embodiments, incorporation of
.sup.18O is not substantially different as compared with the
methods described for the preparation of isotopically labeled
N-substituted piperazine acetic acids in Section II, above. In some
other embodiments, incorporation of .sup.18O is substantially
different and takes advantage of the very caveat that creates some
concern about the methods previously discussed.
[0048] The caveat with respect to the preparation of .sup.18O
labeled N-substituted piperazine acetic acids lies with the
exchange of .sup.18O.sup.16O that can occur between unlabeled water
(H.sub.2.sup.16O) and the .sup.18O of a heavy carboxylic acid
group. A carboxylic acid group is inherently acidic. Acid can
catalyze the exchange of the oxygen atoms of a carboxylic acid
group and water, such as residual water in a sample or water used
in a reaction (e.g. hydrolysis of an ester). Consequently, whenever
.sup.18O labeled N-substituted piperazine acetic acids were
desired, one of two different synthetic routes was chosen.
[0049] In some embodiments, the .sup.18O labeled N-substituted
piperazine acetic acid was obtained by alkylation with an
appropriately .sup.18O labeled halo acetic acid moiety. The
procedure is essentially as outlined in Section II, above except
that an acid labile ester of the halo acetic acid was used in the
alkylation reaction. In some embodiments, the halo acetic acid
moiety comprised the formula:
##STR00011##
wherein Hal is previously defined and R' is an acid labile ester
group, including but not limited to tert-butyldimethylsilyl or
t-boc.
[0050] With reference to FIG. 3A and Example 8, the
tertbutyldimethylsilyl (TBDMS) ester of (.sup.18O).sub.2
bromoacetic acid (14) was used in the alkylation reaction. This
ester was prepared using .sup.18O labeled bromoacetic acid (13),
obtained as a custom order from Cambridge Isotope Laboratory, Inc.,
and TBDMS-CN. The TBDMS ester of N-methyl piperazine acetic acid
(15) was the product of the alkylation with N-methyl piperazine.
The TBDMS ester was selected so that it could be converted to the
acid chloride with, for example, oxalyl chloride thereby avoiding
the requirement for any water and the possible exchange of .sup.18O
with .sup.16O. In the presence of solid phase base (ss-TBD) and
N-hydroxysuccinimide (NHS), the acid chloride was converted to the
NHS ester (16). If the carboxylic acid is desired, instead of the
active ester, the TBDMS ester could be converted to the carboxylic
acid by treatment with an anhydrous acid such as TFA. Accordingly,
aqueous treatment that might lead to .sup.18O.sup.16O exchange, can
be avoided whether the active ester or the carboxylic acid is
desired.
[0051] In some other embodiments, the alkylation to produce
N-substituted piperazine acetic acid proceeded as described in
Section II, above and the .sup.18O was later incorporated. With
reference to FIG. 3B and Example 9, it was found that .sup.18O
could be incorporated into the carboxylic acid group of any
N-substituted piperazine acetic acid by treatment of the
N-substituted piperazine acetic acid with H.sub.2.sup.18O under
acidic conditions. For example and with reference to FIG. 3B, an
isotopically enriched N-methyl piperazine acetic acid (17) lacking
.sup.18O, used to produce the 114 labeling reagent, was treated
with H.sub.2.sup.18O and either HCl or TFA to thereby produce the
TFA or HCl salt of the .sup.18O isotopically enriched N-methyl
piperazine acetic acid (18) and (19).
[0052] Furthermore, the isotopic purity of the product could be
increased by repeated cycles of treatment with H.sub.2.sup.18O
under acidic conditions. The higher the state of enrichment of the
H.sub.2.sup.18O, the fewer cycles required to produce highly
.sup.18O enriched N-substituted piperazine acetic acid. When
H.sub.2.sup.18O of 99% purity was used, the isotopic enrichment of
N-substituted piperazine acetic acid was typically 96% after two
cycles. Because this exchange was performed under acidic
conditions, the product was easily isolated as the bis-acid salt of
N-substituted piperazine acetic acid (e.g. the bis-TFA or bis-HCl
salt).
[0053] Consequently, the present invention contemplates all
possible isotopically enriched N-substituted piperazine acetic
acids comprising one or more heavy atom isotopes of the general
formula:
##STR00012##
including all possible salt forms thereof.
IV. Preparation of Various Active Esters of N-Substituted
Piperazine Acetic Acid
[0054] In some embodiments, this invention pertains to methods for
the preparation of active esters of N-substituted piperazine acetic
acid, including isotopically enriched versions thereof, as well as
the N-substituted piperazine acetic acid esters themselves, and
isotopically enriched versions thereof. The active ester can be any
active ester. In some embodiments, the active ester can be formed
using an alcohol or thiol of the following formula:
##STR00013##
wherein X is O or S, but preferably O. In some other embodiments,
the active ester can be formed using an alcohol or thiol of the
following formula:
##STR00014##
wherein X is O or S, but preferably O.
[0055] In some embodiments, the active ester can be prepared
through an intermediary imidazolide. According to this method, an
N-substituted piperazine acetic acid ester, including isotopically
enriched versions thereof, can be converted to the imidazolide. The
imidazolide so prepared can then be reacted with the alcohol of
choice to thereby produce the active ester of the selected
alcohol.
[0056] With reference to FIG. 4A and Example 10, this procedure was
used to prepare active esters of 2,2,2-trifluorethanol and
1,1,1,3,3,3-hexafluoro-2-propanol. According to the figure and the
example, the phenyl ester of N-methyl piperazine acetic acid (20)
was treated with trimethyl silyl imidizole (TMS-imidizole) and
sodium phenoxide to form the imidazolide of N-methyl piperazine
acetic acid (21). The imidazolide (21) was then reacted with either
2,2,2-trifluorethanol or 1,1,1,3,3,3-hexafluoro-2-propanol to
produce the desired active ester of N-methyl piperazine acetic acid
(22) or (23), respectively as a bis-acid salt.
[0057] In some other embodiments, the active ester can be prepared
by conversion of the N-substituted piperazine acetic acid,
including isotopically enriched versions thereof, to an acid
chloride followed by subsequent reaction of the acid chloride with
the alcohol of choice to thereby produce the active ester of the
selected alcohol.
[0058] With reference to FIG. 4B and Example 11, the preparation of
the NHS and NHP esters of N-methyl piperazine acetic acid are
illustrated using this general procedure. According to the figure
and the example, N-methyl piperazine acetic acid is treated with
oxalyl chloride to produce the acid chloride (24). The acid
chloride is then treated with either of NHP or NHS and solid phase
base to thereby produce the active ester (25) or (26), respectively
as the free piperazine base (not as an acid salt).
[0059] FIG. 4B also illustrates the application of oxalyl chloride
to the production of the pentafluorophenyl (Pfp) ester (27) wherein
a solution phase base (e.g. triethylamine) is used. The reaction
proceeded well with the solution phase base but the hydrochloride
salt of the base proved difficult to remove. Application of the
solid phase base avoids this caveat.
[0060] In still some other embodiments, the active ester can be
prepared by treatment of the N-substituted piperazine acetic acid,
including isotopically enriched versions thereof, with a
trihalooacetate ester of the alcohol that is desired to form the
active ester of the N-substituted piperazine acetic acid. In this
context, halo refers to fluorine, chlorine, bromine and iodine but
preferably to fluorine and chlorine. The trihalooacetate ester has
the general formula:
##STR00015##
wherein Hal refers to a halogen (fluorine, chlorine, bromine or
iodine) and LG refers to the leaving group alcohol. The leaving
group (LG) of the trihaloacetate esters can have the following
general formula:
##STR00016##
wherein X is O or S, but preferably O. Active esters of N-methyl
piperazine acetic acid comprising these leaving groups (LG) were
successfully prepared using the identified trifluoroacetate esters
(where X is O).
[0061] This procedure can be applied to the N-substituted
piperazine acetic acids whether they are the acid salt or the
zwitterion form. The N-substituted piperazine acetic acids can be
reacted with the triholoacetate ester of the alcohol to thereby
produce the active ester of the N-substituted piperazine acetic
acid. A base that can deprotonate the basic nitrogen atoms of
piperazine ring can be added to the reaction as need to induce
formation of the product when the starting material is an acid salt
of N-substituted piperazine acetic acid. The active ester of the
N-substituted piperazine acetic acid can itself be isolated as the
mono-acid salt or the di-acid salt. (e.g. the mono-TFA salt, the
mono HCl salt, the bis-TFA salt or the bis-HCl salt.). When the
trihalooacetate ester is reacted with an N-substituted piperazine
acetic acid the product can be:
##STR00017##
or a salt thereof, wherein X, Y and Z are previously defined. The
group LG is the leaving group of the active ester that is displaced
by the reactive group of an analyte to be labeled; in essence the
leaving group is the alcohol used to form the active ester.
[0062] Certain trihaloacetate esters are commercially available.
For example, the trifluoracetate esters of pentafluorphenol and
4-nitrophenol can be purchased from commercial sources. However,
the others can be obtained by reacting the desired alcohol with
trihaloacetic anhydride. With reference to Table 1, below, the
trifluoroacetate esters of Pcp, Dhbt, NHS, 3-NP and NHP were
prepared by reacting the respective alcohol with trifluoracetic
anhydride. The general procedure for such reactions can be found in
Example 12. Other alcohols that can be used to produce
trihaloacetate esters suitable for the formation of other active
esters can also be used.
[0063] FIG. 4C illustrates the production of the 114 and 115
labeling reagents as the NHS ester. Accordingly, the procedure was
successfully applied to the production of isotopically enriched
active esters of N-substituted piperazine acetic acids. These
active ester reagents were produced as the bis-HCl salts from the
bis-HCl salts of the piperazine base.
[0064] FIG. 4D illustrates the production of numerous other active
esters of N-methyl piperazine acetic acid that were produced using
this generic process. As will be appreciated by the ordinary
practitioner, this procedure is generic and robust and can be
applied to the production of numerous other active esters of a
plethora of N-substituted piperazine acetic acid derivatives.
V. Isotope Incorporation Pathway for the Preparation of a Set of
Isobaric Labeling Reagents
[0065] FIG. 5A illustrates the general pathway taken to the
production of a set of four isobaric labeling reagents identified
as 114, 115, 116 and 117. These designations are based upon the
"signature ion" each reagent produces upon fragmentation in a mass
spectrometer (FIG. 5B). The "signature ion" can be used to
deconvolute information associated with different samples in a
multiplex assay as discussed in the Introduction.
[0066] The pathways illustrated in FIG. 5A utilize the procedures
set forth above for the production of N-substituted piperazine
acetic acids, and active esters thereof. In particular, suitable
isotopically labeled glycines were used in the preparation of
suitable isotopically labeled N-substituted piperazines (e.g.
N-methyl piperazines). The labeled and unlabeled N-methyl
piperazines can be treated with isotopically labeled bromoacetic
acid derivatives, with or without subsequent .sup.18O enrichment to
thereby produce the N-methyl piperazine acetic acid compounds of
desired structure. These suitably labeled N-methyl piperazine
acetic acid compounds were used as labeling reagents; in the
present case by conversion to an active ester for coupling with
analytes such as peptides.
[0067] All four of the labeling reagents (114, 115, 116 and 117)
were produced as NHS esters. All four reagents were used to label
peptides, including peptides (analytes) obtained from digested
protein. The set of reagents (two or more of them), were shown to
be suitable for the multiplex analysis, including proteome
analysis, as described in copending and co-owned application Ser.
No. 60/443,612, incorporated herein by reference.
[0068] For example, two or more samples containing digested
peptides as the analyte, each sample being labeled with one of the
isobaric labeling reagents (114, 115, 116 or 117), were mixed to
form a mixture that was analyzed in a tandem mass spectrometer.
After the first MS analysis, selected ions, of a particular mass
representing a mixture of fragment ions of the same analyte labeled
with two or more different isobaric labels, were subjected to
dissociative energy causing fragmentation of the selected ions. The
selected ions, and the fragments thereof, were then re-analyzed in
the mass spectrometer wherein signature ions of the isobaric
labeling reagents used to label the analytes, as well as daughter
ions of the analyte, were observed.
VI. State Of Isotopic Enrichment
[0069] The various N-substituted piperazines, N-substituted
piperazine acetic acids and active esters of N-substituted
piperazine acetic acid can be prepared with starting materials of
greater than 80 percent isotopic purity of for each heavy atom
isotope. The isotopic purity can be greater than 93 percent for
each heavy atom isotope in some starting materials. In other
starting materials the isotopic purity can be greater than 96
percent for each heavy atom isotope. In still other starting
materials the isotopic purity can be greater than 98 percent for
each heavy atom isotope. When performing an .sup.16O to .sup.18O
exchange, it was possible to routinely obtain carboxylic acid
groups of 96 or greater percent isotopic purity (per oxygen atom)
of the heavy atom isotope.
[0070] Because, with the exception of .sup.18O which can be
exchanged with .sup.16O in certain cases, the isotope purity and
composition of starting materials will translate directly into the
isotopic purity of the products. Moreover, for .sup.18O, it has
been shown that isotopic purity of greater than 96 percent (per
atom) can be achieved using the methods described herein.
Accordingly, in some embodiments, this invention pertains to
N-substituted piperazines, N-substituted piperazine acetic acids
and/or active esters of N-substituted piperazine acetic acid having
an isotopic purity of at least 80 percent for each heavy atom
isotope. In some other embodiments, this invention pertains to
N-substituted piperazines, N-substituted piperazine acetic acids
and/or active esters of N-substituted piperazine acetic acid having
an isotopic purity of at least 93 percent for each heavy atom
isotope. In still some other embodiments, this invention pertains
to N-substituted piperazines, N-substituted piperazine acetic acids
and/or active esters of N-substituted piperazine acetic acid having
an isotopic purity of at least 96 percent for each heavy atom
isotope. In yet some other embodiments, this invention pertains to
N-substituted piperazines, N-substituted piperazine acetic acids
and/or active esters of N-substituted piperazine acetic acid having
an isotopic purity of at least 98 percent for each heavy atom
isotope.
[0071] The following examples are illustrative of the disclosed
compositions and methods, and are not intended to be limit the
scope of the invention. Without departing from the spirit and scope
of the invention, various changes and modifications of the
invention will be clear to one skilled in the art and can be made
to adapt the invention to various uses and conditions. Thus, other
embodiments are encompassed.
EXAMPLES
[0072] General Synthetic Notes: Unless otherwise stated, chemicals
were purchased from commercial sources and used as received. Unless
otherwise stated, the following chemicals were purchased from
Sigma-Aldrich. Trifluoroacetic anhydride (TFAA, P/N 106232),
N-Hydroxysuccinimide (NHS, P/N 13067-2), tert-Butyl bromoacetate
(P/N 124230), 4-Nitrophenyl (4-NP) trifluoroacetate (P/N N22657),
Pentafluorophenyl (Pfp) trifluoroacetate (P/N 377074),
tert-butyldimethylsilyl (TBDMS) cyanide (407852),
1-(Trimethylsilyl)imidazole (P/N 153583), Phenyl bromoacetate (P/N
404276), Pentachlorophenol (Pcp-OH, P/N P2604),
2,2,2-Trifluoroethanol (P/N 326747),
1,1,1,3,3,3-Hexafluoro-2-propanol (HFI--OH, P/N 105228),
(3-Hydroxy-1,2,3-benzotriazin-4(3H)-one (Dhbt-OH, P/N 327964),
Oxalyl chloride (P/N 320420), 1-Methylpiperazine (P/N 130001),
Tetrahydrofuran (THF dry, P/N 186562). Dichloromethane (DCM dry,
P/N 270997), 4 M hydrochloric acid (HCl) solution in dioxane (P/N
345547), HCl (gas, P/N, 295426), 3-Nitrophenol (3-NP--OH, P/N
163031) 1,5,7-Triazabicyclo[4.4.0]dec-5-ene (TBD) bound to
polystyrene crosslinked with 2% DVB, Capacity (base): .about.2.6
mmol/g (ss-TBD, Fluka, P/N 90603), H.sub.2.sup.18O (Isotec, 95
.sup.18O atom % (P/N 329878) or 99% .sup.18O atom % (P/N 487090)),
Br.sup.13CH.sub.2COOEt (Cambridge Isotope Laboratories (CIL), P/N
CLM-1010-5), Br.sup.13CH.sub.2.sup.13COOEt (CIL, P/N CLM-1011-1),
BrCH.sub.2C.sup.18O.sup.18OH (Isotec, P/N 597031). All moisture
sensitive reactions were performed under nitrogen or argon
atmosphere.
[0073] Isotopically enriched starting materials were generally
obtained from either Isotec (a Sigma-Aldrich company) or Cambridge
Isotope Laboratories (Andover, Mass.). Generally, the most highly
enriched starting materials were obtained and used in the
production of the isotopically enriched piperazine derivatives.
However, the state of isotopic enrichment of starting materials is
a choice which the ordinary practitioner will appreciate strikes a
balance between the price of the starting materials (wherein the
higher the state of isotopic enrichment, the higher the price) and
requirement for purity of the enriched isotopes in the final
product. Accordingly, the ordinary practitioner will appreciate
that the most practical method of synthesis of isotopically
enriched compounds may not always proceed through the most common
synthetic routes. Indeed, there may be two or more different routes
to the different isotopic variants of the same compound. Thus, for
some reactions and/or compounds described herein, various synthetic
routes have been undertaken and are therefore discussed below.
Certain advantages and caveats pertaining to these routes are also
discussed.
I. Synthesis of Isotopically Labeled N-Methyl Piperazines
[0074] Note: Unlabeled N-methyl piperazine (a.k.a. 1-methyl
piperazine) is commercially available from a variety of sources.
However, no source for any type of isotopically enriched N-methyl
piperazine (as a stock item) could be found. It was determined
however that suitably protected glycine and sarcosine could be
condensed, cyclized and the product (a diketone) could be reduced
to thereby produce N-methyl piperazine (See FIG. 1). Furthermore,
it was determined that all possible permutations of .sup.15N and
.sup.13C isotopically labeled glycine, as well as partially
protected versions thereof (e.g. t-boc protected amino acids), were
commercially available from sources such as Isotec or Cambridge
Isotope Laboratory, Inc. Accordingly, this appeared to be a
promising route to various N-methyl piperazine compounds comprising
one or more heavy atoms. Because appropriately protected (t-boc)
isotopically enriched glycines and suitably protected sarcosine can
be purchased from commercial sources and because the protection of
amino acids, such as glycine and sarcosine, are well-known, this
discussion of the synthetic route to N-methyl piperazine begins
with the suitably protected amino acids (FIG. 1).
Example 1
General Procedure for the Condensation of Sarcosine Ester and
T-boc-Glycine (FIG. 1)
[0075] Note: Sarcosine is commercially available as either the
methyl or the ethyl ester. Either can be used in the condensation
reaction.
[0076] To a round bottom flask (RBF) was added 1.1 equivalent (eq.)
of sarcosine ethyl ester (2) and 1 eq. of t-boc-glycine (1)
(including isotopically labeled t-boc-glycines for the production
of various isotopically labeled N-methyl piperazines). The solid
was then dissolved with the addition of dichloromethane (DCM)
(.about.20 mL/g of t-boc-glycine). To this stirring solution was
added 1.1 eq. of N-methyl morpholine (NMM) then 1.1 eq. of
dicyclohexylcarbodiimide (DCC) in DCM. A precipitate formed within
minutes. The reaction was stirred overnight. The reaction was
monitored by thin layer chromatography (TLC). If t-boc-glycine was
still present, additional DCC in DCM was added. When the reaction
was determined to be complete, the solids were filtered off and the
cake was rinsed with DCM. The product containing solution was then
evaporated to dryness.
[0077] The product was purified by silica gel chromatography using
a column packed in 50% ethyl acetate (EtOAc)/hexane. A small amount
of the 50% EtOAc/hexane solution was used to dissolve/suspend the
dried down product (not all will dissolve). This solution/slurry
was loaded onto the packed column. The column was eluted 50%
EtOAc/hexane to obtain the minimally retained product. Product
containing fractions were evaporated to provide an oil, speckled
oil, or flaky solid (materials that are higher in heavy atom
isotope content appeared to exhibit more characteristics of a
solid).
TABLE-US-00001 TLC conditions: EtOAc (developed with ninhydrin and
heat) Product (3) Rf~0.85 t-boc-glycine (1) Rf~0.3 (broad tailing)
Sarcosine-OEt (2) Baseline NMM Faintly visible just above
sarcosine
Example 2
General Procedure for the Synthesis of
1-Methyl-2,5-Diketopiperazine (FIG. 1)
[0078] A solution of 1:1 trifluoroactetic acid (TFA):DCM containing
0.5% water was prepared. This solution was added to the column
purified product (3) of the condensation reaction (.about.10 mL/g
starting material). The resulting solution was stirred for 30
minutes and then the solvents removed by rotoevaporator. Ethanol
(.about.10 mL/g starting material) was then added to the reaction
flask and this solution was again striped to dryness. The procedure
was repeated with toluene. The product was again dissolved in
ethanol in the reaction flask and anhydrous potassium carbonate (4
eq) was added. The solution bubbled vigorously for a short period
following the addition of the potassium carbonate. A drop of the
reaction mixture was removed, diluted with water, and the pH of the
solution was determined. If the pH was below 8, more potassium
carbonate was added. Once the pH was confirmed to be greater than
8, the reaction was allowed to reflux overnight. The warm reaction
mixture was then passed through a plug of Celite to remove the
excess salts. The cake was rinsed twice with anhydrous ethanol. The
filtrate was transferred to a larger flask and stripped to dryness.
The product foam was redissolved in 9:1 ethyl acetate-methanol and
passed through a plug of silica-gel. The silica-gel was then washed
with .about.4 column volumes of 9:1 ethyl acetate-methanol. All
fractions were evaporated to dryness.
[0079] Notes and alternative procedures: Deprotection of the t-boc
group with the TFA/DCM/H.sub.2O solution can be followed by TLC
(ninhydrin/heat shows conversion of the spot at Rf 0.85 to a dark
red-brown spot at origin). After deprotection, it is also
acceptable to add methanol, concentrate and re-treat with methanol
followed by a second concentration and drying in vacuo to remove
excess TFA.
[0080] In some reactions, concentrated ammonium hydroxide (large
excess .about.60 mL per 16 mmol of starting material) was
substituted for potassium carbonate. (When concentrated ammonium
hydroxide was added to the reaction at room temperature, it
generated a white insoluble material and a slightly milky reaction
mixture.) After the addition of concentrated ammonium hydroxide,
the flask was sealed with septum to prevent loss of ammonia.
Cyclization appeared to be complete after overnight (12 hrs)
reaction although in some cases heating to 60.degree. C. over
several hours was sufficient. The reaction was monitored by TLC
(10% MeOH/DCM visualized with 10% phosphomolybdic acid (PMA) in
MeOH with heat). The product appeared as a blue spot at Rf 0.54.
Since the deblocked material (red-brown spot at origin) could not
be visualized with PMA, another TLC was performed as a
cross-reference using ninhydrin/heat.
[0081] When cyclization was deemed complete by TLC analysis, the
mixture was filtered and the flask and solids were rinsed with DCM.
The filtrate was concentrated and redissolved in 10% MeOH/DCM
before chromatography. The white waxy solids were partially
insoluble in the 10% MeOH/DCM so the material was sonicated.
Sonification successfully dissolved the mixture that was then
applied to the column. The first fraction eluted was mainly the
white waxy solid. The major fraction (the dione product (4)) eluted
next and was followed by another minor impurity (Rf 0.3). It was
observed that in cases where incomplete TFA removal resulted in
formation of ammonium triflate, this impurity co-eluted with the
product and the secondary material. A second column could be used
to completely purify the desired product.
[0082] The melting points of the dione (4): 116, 117: 138-139 for
model compound: lit: 136-139 (J. Het. Chem 18, 423, 1981); 142-143
(J. Biol. Chem 61, 445, 1924).
[0083] TLC condition: 9:1 ethyl acetate-methanol (develop with
phosphomolybdic acid and heat)
[0084] Product Rf.about.0.2
[0085] Alternative TLC condition: 10% MeOH/DCM; develop with heat
Product (blue spot) Rf=0.54
[0086] 1H-NMR Data
##STR00018##
[0087] NMR (D.sub.6 DMSO)--N1-CH.sub.3, d 2.79, 2.80 3H; 3-CH.sub.2
dd 3.94, 3.92, 3.59, 3.57 2H; 6-CH.sub.2 d 3.87, 3.86 2H; 4-NH b
8.11H.
##STR00019##
[0088] NMR (D.sub.2O)--N1-CH.sub.3, d 3.00, 2.99 3H; 3-CH.sub.2 d
4.08, 4.08 2H, 6-CH.sub.2 d 4.14, 4.14 2H.
##STR00020##
[0089] NMR (D.sub.6 DMSO)--N1-CH.sub.3, s 2.80 3H; 3-CH.sub.2 s
3.76 2H; 6-CH.sub.2 s 3.86 2H; 4-NH d 8.03, 8.26 1H.
Example 3
General Procedure for the Synthesis of N-Methyl Piperazine (Route
A; FIG. 1)
[0090] A saturated solution of sodium sulfate was prepared.
Tetrahydrofuran (THF) (4 mL per mmol starting material based upon
the material used in Example 2) was added to the diketopiperazine
formed using the procedure of Example 2. The reaction flask was fit
with a reflux condenser and three equivalents of 1M LiAlH.sub.4 in
THF (LAH solution; it may be possible to substitute Red-Al or other
reducing reagent for LAH but this has not been attempted) was added
to the solution through a dropping funnel. There was vigorous
hydrogen evolution at the initiation of the addition but this
subsided as the addition continued. The reaction was heated to
reflux for 4 hours. After the reaction was complete, the solution
was cooled to room temperature and the remaining LAH was quenched
with the very slow addition of saturated aqueous sodium sulfate
(1/4 the volume of the LAH solution added). The reaction appeared
as a gray suspension.
[0091] DCM was added to this suspension (1/2 volume of the THF) and
the gray gel-like solid was removed by filtration. The flask and
filtered solids were then thoroughly washed 2.times. with DCM (1/4
volume of the THF). The combined organic solution (DCM/THF) was
then dried with Na.sub.2SO.sub.4 (solid--anhydrous) and filtered.
(In some early experiments the N-methyl piperazine was isolated as
an oil (free base and not as a TFA or HCl salt) but the product was
determined to be a volatile oil and therefore not be isolated in
high yield).
[0092] Di-tert-butyl-dicarbonate (3 equivalents) was added to this
solution that was stirred and vented overnight. TLC was used to
monitor the reaction. Once complete, the solvent was removed by
rotary evaporation to yield a liquid. This liquid is slightly
volatile, so low vacuum evaporation of solvent is recommended (high
vacuum conditions should be avoided). The product was dissolved in
DCM and loaded onto a silica-gel column packed with 8% methanol in
ethyl acetate. Product was eluted with the 8% methanol in ethyl
acetate solution. Product containing fractions were determined by
TLC, pooled, and evaporated to a liquid. This liquid was taken
directly to the deprotection reaction. Note: the t-boc deprotection
was performed only as a means to isolate the crude N-methyl
piperazine product but this requires subsequent deprotection.
TLC-N-methyl piperazine (develop with ninhydrin) 4:1:1
Ethanol:Water:Ammonium hydroxide
Product Rf=0.6
[0093] TLC-N.sup.1-t-Boc-N.sup.2-methyl piperazine (develop with
ninhydrin)
4:1 DCM-MeOH
Product Rf=0.5
Deprotection:
[0094] A solution of 1:1 TFA, DCM with 0.5% water was prepared.
This solution was added to the material isolated from the
reduction, above (.about.10 mL/g starting material). The reaction
was stirred for 30 minutes then the solvent was removed with a
rotoevaporator. Solvent evaporation was terminated when no more
solvent was observed to be collecting on the condenser. TFA was
added to the product residue (.about.2 mL/g starting material) to
form a free flowing solution. The TFA solution was transferred to a
centrifuge tube and diethyl ether was added to precipitate the
product salt. The solution was mixed using a vortex. The solution
was then centrifuged and the supernatant decanted to collect the
precipitate. The filtrate was then washed 1 time with ether by
resuspending the product, vortexing, and re-centrifugation. Product
was dried under low vacuum to remove residual ether.
[0095] 1H-NMR Data:
##STR00021##
[0096] NMR (D.sub.2O)--N1-CH.sub.3, d 3.02, 3.03 3H; methylenes,
broad triplet 3.3-3.9 8H
##STR00022##
[0097] NMR (D.sub.2O)--N1-CH.sub.3, d 3.02, 3.03 3H; methylenes,
broad 3.40-3.85 8H
##STR00023##
[0098] NMR (D.sub.2O)--N1-CH.sub.3, s 3.03 3H; methylenes, broad
3.50-3.75 8H
Example 4
General Procedure for the Synthesis of N-Methyl Piperazine (Route
B; FIG. 1)
[0099] The product of the procedure of Example 2 was dissolved in
anhydrous THF (5 mL per mmol SM) in a multi-neck RBF fitted with
condenser, addition funnel and argon (Ar) inlet. To this solution
was added 3 equivalent of the LAH solution slowly through a
dropping funnel at RT under Ar. Vigorous hydrogen evolution was
observed at the beginning. After addition, the cloudy solution was
heated to reflux for 3 hours. TLC was used to determine when the
reaction was complete (disappearance of starting material (SM), 10%
MeOH/DCM TLC developing solvent, PMA as visualizer). After the
reaction was complete, the solution was cooled to room temperature
and quenched with the very slow addition of saturated aqueous
sodium sulfate (1/4 the volume of the LAH solution added). White
gel-like solid solution was passed through a plug of
Na.sub.2SO.sub.4 solid to remove H.sub.2O. The filter cake was
washed with THF several times (400 mL per gram SM) until TLC of the
washing showed a little product. Then TFA (4 eq) was added to the
THF solution (HCl in dioxane could also be added if the HCl salt
was desired). The color of the solution changed to light brown from
pale yellow. The solution was concentrated on a rotoevaporator
under reduced pressure to yield brown oil. The light brown product
was precipitated as bis-TFA salt by adding ether (42 mL per 1 gram
SM) to yield of 80% N-methyl-piperazine. .sup.1H NMR (D.sub.2O) was
used to confirm the desired product.
II. Alkylation of N-Methyl Piperazines to Form N-Methyl Piperazine
Acetic Acids
[0100] Note: FIG. 5A illustrates the pathway for the synthetic
incorporation of heavy atom isotopes into four isobaric labeling
reagents referred to herein as 114, 115, 116 and 117. As can be
seen from FIG. 5A, certain of the heavy atom isotopes can be
incorporated by the choice of the commercially available
isotopically labeled glycine used in the production of the N-methyl
piperazine. Certain other heavy atoms can be incorporated during
the alkylation reaction based upon the choice of the commercially
available bromoacetic acid. In some cases, the .sup.18O can be
incorporated through an efficient exchange using .sup.18O labeled
water. The labeling reagents are designated 114, 115, 116 and 117
based upon the mass of the fragment that forms a signature ion in
the mass spectrometer (see: FIG. 5A and FIG. 5B) once the reagent
has been fragmented by the application of dissociative energy.
[0101] With reference to FIG. 2A, scheme A is useful for producing
the N-methyl piperazine acetic acid as a zwitterion and not as a
salt (e.g. mono or bis TFA or HCl salt). With reference to FIG. 2B,
scheme B is useful since it requires the use of only one equivalent
of N-methyl piperazine for the production of the N-methyl
piperazine acetic acid thereby foreclosing the waste of the
valuable isotopically labeled starting material. With reference to
FIG. 2C, scheme C is useful for alkylations involving the
isotopically labeled bromoacetic acid, particularly the .sup.18O
labeled bromoacetic acid as it was expected to reduce the
occurrence of .sup.18O scrambling (or exchange with .sup.16O from
residual water).
Example 5
Procedure for the Synthesis of Isotopically Labeled N-Methyl
Piperazine Acetic Acids (Scheme A; FIG. 2A)
[0102] To a stirring solution of 1.18 g (11.83 mmol) N-methyl
piperazine in 15 mL of toluene at room temperature was added 1 g
(5.91 mmol) of ethylbromoacetate,1,2-.sup.13C dropwise, over a
period of 15 minutes. Immediate formation of white solid was
observed. The reaction mixture was then heated in an oil bath at
90.degree. C. for 4 hr. After cooling the mixture to room
temperature, the off-white solid was removed by filtration, and
washed with 25 mL of toluene. The combined filtrate and washings
was then concentrated in a rotoevaporator, and the residue was
dried under high vacuum for 5 hours to yield 1.10 g (quantitative)
of ethyl ester of N-methyl piperazine acetic acid-1,2-.sup.13C (9)
as an off-white oil. The crude product (9) was analyzed by MS and
.sup.1H-NMR, and was directly used for the next step without
further purification. MS (ESI, m/z): 189.16 (M+1), .sup.1H-NMR
(DMSOd.sub.6) .quadrature..quadrature.4.2 (m, 2H), 3.4 (d, 1H, J=7
Hz), 3.05 (d, 1H, J=7 Hz)), 2.4-2.7 (b, 8H), 2.3 (s, 3H), 1.25 (t,
3H).
[0103] A solution of ethyl ester of N-methyl piperazine acetic acid
(9) (1.1 g, mmol), prepared as described above, in water (20 mL)
was refluxed for 24 hr. The reaction was monitored by MS analysis.
After 24 hr, the reaction mixture was concentrated in a
rotoevaporator to afford white solid product, which was triturated
with acetone (10 mL) overnight. The product was then separated by
filtration and dried under high vacuum overnight at 45.degree. C.
in a vacuum oven, to yield 780 mg of N-methyl piperazine acetic
acid, 1, 2-.sup.13C (10), as a white powdery solid. 300 mg of the
product was further purified by sublimation (1 mm/Hg,
110-120.degree. C.) to yield 270 mg of white solid. MS (ESI, m/z);
161 (M+1), .sup.1H-NMR (DMSOd.sub.6) 3.3 (d, 1H, J=7 Hz), 2.95 (d,
1H, J=7 Hz), 2.55-2.75 (b, 4H), 2.3-2.45 (b, 4H), 2.18 (s, 3H)
[0104] Notes: This procedure utilizes unlabeled N-methyl
piperazine. This procedure is useful for producing the zwitterion
of N-methyl piperazine acetic acid.
[0105] The product can also be isolated as the mono or bis-HCl or
mono or bis-TFA salt by treatment with the appropriate acid prior
to or subsequent to its isolation as described above.
Example 6
Procedure for the Synthesis of Isotopically Labeled N-Methyl
Piperazine Acetic Acids (Scheme B; FIG. 2B)
[0106] To a slurry of 200 mg (1.14 mmol) of
N-methylpiperazine-.sup.15N.2HCl (the .about.2TFA salt can also be
used) in methanol (MeOH, 14 mL), was added 1.76 g (4.59 mmol) of
ss-TBD, with a loading of 2.6 mmol/g, followed by CH.sub.2Cl.sub.2
(6 mL). The mixture was then sonicated for 15 minutes and was then
cooled in an ice bath under an argon atmosphere. To this vigorously
stirred slurry, a solution of 193 mg (1.14 mmol) of
ethylbromoacetate-2-.sup.13C in acetonitrile (3 mL) was added
dropwise using a syringe pump (maintaining a rate of 2 mL/hr).
After completion of the addition, the ice bath was removed and the
mixture was continued stirring at room temperature overnight (18
hr). The mixture was then filtered through a sintered funnel, and
the solid was washed several times with MeOH (4.times.10 mL). The
combined filtrate and washings were then concentrated in a
rotoevaporator, and the residue was kept under high vacuum to yield
111 mg (51%) of the ethyl ester of the N-methyl piperazine acetic
acid (11) as an off white solid. This crude product was directly
used for the next step without further purification. MS (ESI, m/z)
189 (M+1). .sup.1H-NMR (DMSOd.sub.6) 4.05 (q, 2H), 3.3 (s, 1H), 3.0
(s, 1H), 2.4-2.5 (b, 4H), 2.2-2.4 (b, 4H), 2.1 (s, 3H), 1.15 (t,
3H).
[0107] The product was hydrolyzed in the manner described in Scheme
A, above. The following analytical data was obtained for the
product.
[0108] MS (ESI, m/z) 161 (M+1). .sup.1H-NMR (DMSOd.sub.6) 3.35 (s,
1H), 3.05 (s, 1H), 2.65-2.8 (b, 4H), 2.5-2.65 (b, 4H), 2.35 (s,
3H),
[0109] Without substantial variation, above general procedure was
applied to other isotopically labeled N-methyl piperazines to
produce various isotopically labeled N-methyl piperazine acetic
acid derivatives.
[0110] The product can also be isolated as the bis-HCl or bis-TFA
salt by treatment with the appropriate acid prior to or subsequent
to its isolation as described above.
Example 7
General Procedure for the Synthesis of Isotopically Labeled
N-Methyl Piperazine Acetic Acids (Scheme C; FIG. 2C)
[0111] To a solution of bromoacetic acid (715 mg, 5 mmol) in DCM
(15 mL) was added 700 mg of trityl-Cl resin (1 mmol, 1.45 mmol/g)
followed by diisopropylethylamine (DIPEA) (1.79 mL, 10 mmol). This
solution was mixed at room temperature for 1 hour. The resin was
then filtered and washed with dichloromethane (3.times.4 mL)
followed by a wash with a solution of
dichloromethane-methanol-DIPEA (17:2:3, 5 mL) and finally a wash
with dichloromethane (3.times.4 mL).
[0112] The resin was then treated with a solution of N-methyl
piperazine (N-MP) (0.57 mL, 5 mmol) in DMF (5 mL) for 30 minutes
and then washed with DMF and dichloromethane (3.times.4 mL each).
The N-MPA so generated on resin was cleaved with a 25% solution of
TFA in dichloromethane (10 mL for 5 min)) and resin was washed with
the same solution (2.times.5 mL). After evaporation of TFA, the
product was precipitated and washed with ether (388 mg, 99% yield,
bis TFA salt). The product was identified by NMR (matched with
literature) and with ES MS (Calculated MH.sup.+=159.11, found
159.14).
[0113] Without substantial variation, the above general procedure
was applied to other isotopically labeled N-methyl piperazines to
produce various isotopically labeled N-methyl piperazine acetic
acid derivatives.
[0114] The product could also be isolated as its bis-HCl salt if
HCl was used to cleave the product from the support rather than
TFA. Other acids could also be used for the cleavage reaction and
product would be the salt of the acid used.
III. Methods for the Incorporation of .sup.18O into N-Methyl
Piperazine Acetic Acids
[0115] Note: In the initial experiments, incorporation of .sup.18O
into the N-methyl piperazine acetic acid was attempted as
illustrated in FIG. 3A using .sup.18O labeled bromoacetic acid
(custom synthesized by CIL). Caveats to this approach include the
possibility that in subsequent reactions, the .sup.18O can exchange
with .sup.16O from residual water or can otherwise exchange with
.sup.16O from other reagents during the esterification process. The
more recently applied synthetic procedure is illustrated in FIG. 3B
and capitalizes on the .sup.16O.sup.18O exchange reaction, using
H.sub.2.sup.18O to drive the equilibrium reaction to formation of
the desired heavy version of the N-methyl piperazine acetic acid.
Though both schemes have been shown to work, Scheme B currently
supports the production of the most highly .sup.18O enriched
products.
Example 8
General Procedure for the Synthesis of .sup.18O Isotopically
Labeled N-Methyl Piperazine Acetic Acids, Including Conversion to
the Active Ester (Scheme A; FIG. 3A)
[0116] To a solution of TBDMS-CN (172 mg, 1.190 mmol)) in DCM
(0.575 mL) was added .sup.18O labeled bromoacetic acid (13) (170
mg, 1.189 mmol) under an argon atmosphere and the solution was
heated to 80.degree. C. for 20 minutes and then cooled to room
temperature. The product (14) was isolated as an oil (254 mg, 85%
yield). .sup.1H NMR (CDCl.sub.3) 3.58 (2H, --CH.sub.2--), 0.955
(9H, (CH.sub.3).sub.3--Si), 0.30 (6H, CH.sub.3Si).
[0117] A solution of BrCH.sub.2C.sup.18O.sub.2-TBDMS (14) (254 mg,
1 mmol) in DCM (2.5 mL) was added (34 .mu.L/min) to an argon
flushed flask containing N-MP (110 .mu.L, 1 mmol), TBD resin (576
mg, 1.5 mmol, 2.6 mmol/g) and DCM at 0.degree. C. After the
addition was complete the reaction continued for 1 h at RT and then
the resin was filtered and washed with DCM. Combined filtrate was
concentrated by rotary evaporation to obtain 118 mg (42% yield) of
an oil (15).
[0118] Note: Because of the potential for .sup.18O .sup.16O
exchange during the esterification, the N-methyl piperazine acetic
acid prepared by this route was not converted to the active ester
using the trifluoracetate procedure described in Section VI, below.
Instead it was converted using oxalyl chloride and NHS as described
below.
[0119] To a solution of .sup.18O containing TBDMS ester of N-MPA
(15) as obtained above (118 mg, 0.427 mmol) in DCM (5 mL) was added
a solution of oxalyl chloride (0.427 mL, 0.854 mmol, 2 M solution
in dichloromethane) at room temperature. The reaction allowed to
continued for 1 hour when an off white slurry formed. Solvent and
excess reagent were removed from the reaction mixture. A solution
of NHS (50 mg, 0.427 mmol) in dry THF (1.4 mL) was added to the
resulting solid followed by 5 mL of dichloromethane, 4 mL of THF
and 246 mg of ss-TBD resin (0.640 mmol, 2.6 mmol/g). The mixture
was sonicated and mixed for 20 minutes, after which the resin was
filtered and washed with 5 mL of dry dichloromethane. To the
filtrate so obtained was added 2 mL of 4.0 M solution of HCl in
dioxane and the precipitate (16) was washed with dry THF (5
mL.times.2) and hexanes (5 mL) and dried under vacuum (10 mg, 7%
yield). ES-MS (direct infusion in i-propanol) shows isotopic purity
to be around 74% at this stage.
Example 9
General Procedure for the Synthesis of .sup.18O Isotopically
Labeled N-Methyl Piperazine Acetic Acids (Scheme B; FIG. 3B)
[0120] 200 mg (1.24 mmol) of N-methyl piperazine acetic acid
1,2-.sup.13C (17) was weighed out in a 5 mL plastic vial flushed
with argon. The vial was then transferred into a glove box and 2.5
mL of .sup.18O-water (>99% .sup.18O) was added. The vial was
then fitted with a silicone septum, and a low stream of HCl gas was
then passed through the solution using a long needle after venting
the septum with an open needle. When the solution had warmed
(.about.2 min), the HCl passage was stopped, and the septum was
replaced with a screw-cap. The vial was then heated at 80.degree.
C. in a heating block for 18 hr. An aliquot was analyzed by MS and
.sup.18O purity was calculated as 93%. The reaction mixture was
then concentrated to dryness in a speedvac, and the residue was
subjected to a second cycle of .sup.18O-exchange as described
above. By MS analysis the .sup.18O purity after the second cycle
was determined as 96%. The mixture was then evaporated to dryness
in a speedvac, and traces of water were removed by co-evaporataion
with toluene (1 mL.times.2). 220 mg of N-methyl piperazine acetic
acid-1,2-.sup.13C -.sup.18O.sub.2.2HCl (18) was obtained. MS (ESI,
m/z), 165 (M+1)
[0121] Note: The product was used without further purification in
the production of active ester of the N-methyl piperazine acetic
acid. The bis-TFA salt was also produced using the above-described
procedure wherein TFA was substituted for HCl.
IV. Preparation of the Active Esters of the N-Methyl Piperazine
Acetic Acids
[0122] Note: Several methods were employed for the production of
active esters of N-methyl piperazine acetic acid. The procedure
illustrated by Scheme A (FIG. 4A) worked well for the production of
the fluoroalcohol esters of N-methyl piperazine (See: FIGS. 4C and
4D). The procedure illustrated by Scheme B (FIG. 4B) produced
various active esters of N-methyl piperazine but unless the solid
phase base was used (e.g. ss-TBD), the hydrochloride salt of
solution phase base was difficult to remove. The procedure
illustrated by Scheme C (FIGS. 4C and 4D) proved to be the most
generally applicable route to the production of active esters of
N-methyl piperazine.
Example 10
Synthesis of Active Esters of N-Methyl Piperazine Acetic Acid Via
Imidazolide Formation (Scheme A, FIG. 4A)
[0123] To a solution of N-methyl piperazine phenyl ester (20) (100
mg, 0.426 mmol) and sodium phenoxide (1 mg, 9 .mu.mol) in THF (5
mL) was added TMS-imidazole (69 .mu.L, 0.468 mmol). The solution
was mixed for 20 minutes to generate the imidazolide (21).
CF.sub.3CH.sub.2OH (80 .mu.L, 0.213 mmol) was then added to the
light yellow solution so obtained. The solution was mixed for
another 30 minutes when TLC indicated clean formation of product
(R.sub.f=0.6, 4:1 DCM-MeOH). The reaction was then diluted to 15 mL
with EtOAc and the product (22) was precipitated by addition of HCl
solution in dioxane (4 M, 2 mL). After washing with THF (2.times.15
mL) product was isolated as white solid. NMR of the solid indicated
a 1:1 mixture of product and imidazole (as HCl salt). Calculated
MH.sup.+=241.13, found=241.12.
[0124] 1,1,1,3,3,3-Hexafluoro-2-propanol ester (23) was isolated
using the general procedure set forth above provided however that
(CF.sub.3).sub.2CHOH was substituted for CF.sub.3CH.sub.2OH. The
following analytical data was obtained for this product.
(R.sub.f=0.37, 9:1 DCM-MeOH). Calculated MH.sup.+=309.11,
found=309.11.
[0125] Note: N-methyl piperazine phenyl ester was prepared by the
alkylation procedures described above (See FIGS. 2A and 2B) wherein
phenyl bromoacetate is substituted for ethyl bromoacetate.
Example 11
Synthesis of Active Esters of N-Methyl Piperazine Acetic Acid Via
Oxalyl Chloride (Scheme B, FIG. 4B)
[0126] To a suspension of N-methyl piperazine acetic acid (N-MPAA)
(79 mg, 0.5 mmol) in DCM (25 mL) was added a solution of oxalyl
chloride (4 mL, 0.8 mmol, 2.0 M solution in DCM) over 10 minute at
room temperature. After another 30 minutes of reaction, solvent and
excess reagent were removed under reduced pressure to give a white
solid (24). A solution of NHS (57 mg, 0.5 mmol) in DCM (25 mL) was
added to the solid followed by ss-TBD (390 mg, 1 mmol, 2.6 mmol/g).
The resulting solution was sonicated for 5 minute when all solid
dissolved. The ss-TBD resin was removed by filtration and solvent
was evaporated to yield a white foam (97% yield). Product was
characterized by ES-MS as before.
Synthesis of Active Esters of N-Methyl Piperazine Acetic Acid Via
Trifluoroacetate Esters (Scheme C, FIGS. 4C and 4D)
[0127] Note: Conversion of the N-methyl piperazine acetic acids
(N-MPAAs) to their active esters via the trifluoracetate ester is
typically a two-step process. Except for the rare case where the
reagent is commercially available (See: Table 1), the first step
involves the preparation of a reagent for esterifying the acetic
acid. The second step involves reacting the esterifying reagent
with the N-methyl piperazine acetic acid to produce the active
ester. Various active esters were produced and tested for the
aqueous labeling of peptides. Though the NHS ester proved to be
quite useful for this application, other esters may prove useful in
other applications. Nevertheless, this method of producing the
active esters proved to be quite robust and generally applicable
across a wide variety of compounds. FIG. 4B illustrates 7 different
active esters that were produced using the same generic
procedure.
Example 12
Synthesis of N-Hydroxysuccinimide Trifluoroacetate.sup.10,11 and
Other Trifluoroacetate Esters
[0128] Trifluoroacetic anhydride (4.9 mL, 4.times.8.68 mmol (2.5-4
equivalents is typically used) was added to N-hydroxysuccinimide
(NHS) (1 g, 8.69 mmol) and stirred under argon for 1-2 h to produce
a homogeneous reaction mixture. Excess reagent and by-product
CF.sub.3COOH were removed under reduced pressure (rotary
evaporation). The product was obtained as white solid in
quantitative yield. The solid was dried under high vacuum for 3-4 h
and stored under argon (Ar) or nitrogen (N.sub.2) gas.
[0129] With reference to FIG. 4D and Table 1, the trifluoroacetate
ester of pentafluorophenol (Pfp) and 4-nitrophenol (4-NP) were
commercially available. The remaining trifluoroacetate esters were
synthesized using the above-described generic procedure provided
however that the reaction time and temperature were varied.
Furthermore, in some cases the products were isolated by
distillation. Yields of the trifluoroacetate esters were good and
in some cases near quantitative. The specific conditions used are
set forth in Table 1, below.
TABLE-US-00002 TABLE 1 PK.sub.a 4.68 ##STR00024## Pcp 5.50
##STR00025## Pfp 7.23 ##STR00026## 4-NP 7.78 ##STR00027## Dhbt 7.80
##STR00028## NHS 8.33 ##STR00029## 3-NP 9.38 ##STR00030## NHP
Example 13
General Method for the Preparation of Active Esters of
N-Substituted Piperazine Acetic Acid from Trifluoroacetate
Esters
[0130] A solution of the trifluoroacetate in THF (0.58 M, 1.2
equiv) was added to a solid sample of N-methyl piperazine acetic
acid and mixed in a vortex or shaker until a homogeneous solution
was obtained. The reaction of the carboxylic acid with the
trifluoroacetate ester was generally complete within 30 min for all
cases except N-hydroypyrrolidinone (NHP, 18 h). The progress of
conversion to the active ester was monitored by ES-MS. The amount
of product and any starting material (N-MPA) could be determined by
direct infusion of a sample of the reaction (in ethanol) into the
ES-MS. In some cases the active ester product was precipitated as
dihydrochloride salt by the addition of a solution by addition of
HCl solution in dioxane (4 M, 50% volume of the reaction) followed
by washing with THF, ethyl acetate and hexanes. In other cases the
product was isolated from the reaction as the mono TFA salt.
Addition of TFA could be performed if the bis-TFA salt was
desired.
[0131] Dhbt ester, Calculated MH.sup.+=304.14 Found=304.20
[0132] NHP ester, Calculated MH.sup.+=242.15 Found=242.20
[0133] 4-NP ester, Calculated MH.sup.+=280.13 Found=280.20
[0134] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.20 (d, 2H, J=9.2
Hz, aromatic protons), 7.25 (d, 2H, J=9.2 Hz, aromatic protons),
3.69-3.40 (broad, 2H, ring protons), 3.57 (s, 2H,
--CH.sub.2--CO--), 3.15-2.90 (broad, 6H, ring protons), 2.78 (s,
3H, --CH.sub.3).
[0135] Pfp ester, Calculated MH.sup.+=325.10 Found=325.10
[0136] Pcp ester, Calculated MH.sup.+=404.95 Found=405.90
[0137] 3-NP ester, Calculated MH.sup.+=280.13 Found=280.20
[0138] NHS ester, Calculated MH.sup.+=256.13 Found=256.10
Example 14
Synthesis of the NHS-Ester of N-Methyl Piperazine Acetic
Acid-1,2-.sup.13C --.sup.18O.sub.2, 2.HCl (the 114 Labeling
Reagent)
[0139] To a slurry of N-methyl piperazine acetic acid
-1,2-.sup.13C, .sup.18O, 2.HCl (28) (60 mg, 0.25 mmol) in THF (1.8
mL), was added DIPEA (98 mg, 0.76 mmol) under argon. The mixture
was vortexed for 5 min, and the trifluoroacetate of
N-hydroxysuccinimide (160 mg, 0.76 mmol) was added. After
sonicating for 10 minutes, the reaction mixture was stirred at room
temperature for 4 hours, followed by a centrifugation to remove any
undissolved material. The supernatant was decanted then diluted
with THF (3 mL) and added slowly to a 4M solution of HCl in dioxane
(1.8 mL). The precipitated HCl salt of the NHS-ester was separated
by centrifugation, and washed with THF (3 mL.times.4), dried under
high vacuum to yield 62 mg (74%) of the NHS ester (30) as an
off-white solid. MS (ESI, m/z) 261 (M+1), .sup.1H-NMR (DMSOd.sub.6)
4.05 (d, 1H, J=7 Hz), 3.7 (d, 1H, J=7 Hz), 3.3-3.45 (b, 2H),
2.95-3.1 (b, 2H), 2.85 (s, 3H), 2.75 (m, 4H).
[0140] With the exception of using a different isotopically
enriched N-methyl piperazine acetic acid, the above describe
procedure was followed for the production of the 115 labeling
reagent (31). The analytical data for the product (31) is as
follows.
[0141] MS (ESI, m/z) 261 (M+1). .sup.1H-NMR (DMSOd.sub.6) 4.05 (s,
1H), 3.7 (s, 1H), 3.3-3.4 (b, 2H), 3.1-2.95 (b, 4H), 2.85 (s, 3H),
2.75-2.80 (b, 1H), 2.7 (m, 4H).
[0142] Notes: The trifluoroacetate ester reagent can be reacted
with the zwitterion of N-methyl piperazine acetic acid and well as
with a mono salt or bis salt (e.g. mono-HCl salt, mono-TFA salt,
bis-HCl salt or bis-TFA salt) of the N-methyl piperazine acetic
acid. When the bis-HCl salt was used a base such as
diisoproplyethylamine (DIPEA) was added to neutralize the acid. The
transesterification reaction did however apparently proceed with
the bis-TFA salt of N-methyl piperazine acetic acid without the
addition of base.
[0143] The examples set forth above are for illustrative purposes
only and should not be viewed as a limitation on the scope of the
invention.
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