Patent Application
20160263256
The present invention generally relates to the field of Magnetic Resonance Imaging (MRI). More particularly, the invention relates to a process using the Para Hydrogen Induced Polarisation (PHIP) technique for the preparation of [1-13C]-hyperpolarized carboxylate compounds of diagnostic interest as MR metabolic probes.
Magnetic Resonance Imaging is a well established powerful tool for medical and biological investigations both in vitro and in vivo. The main drawback of this technique is due to the intrinsic low sensitivity of the NMR spectroscopy on which MRI is based. In fact, the intensity of NMR signals depends on the difference between the nuclear spin states populations of the imaging nuclei. According to the well known Boltzman equation (ΔN=γhB0/(2πkT)), this difference is a function of temperature and applied magnetic field, and, at thermal equilibrium, it is in the order of 10−5, i.e. very low.
The use of hyperpolarized molecules has been recently proposed as a possible solution of the said drawback and, in recent years, many efforts have been devoted to the development of both feasible and effective MR-hyperpolarization procedures.
Driving force in the recorded development stems in the potential this technique offer to overcome the sensitivity limitations of conventional MR Imaging opening a number of innovative applications both in chemistry and, especially, in biology.
Indeed, the significant improvement of the signal strength enabled by this technique for detectable compounds representing key molecules in the metabolic processes led to the development of innovative MR procedures exploiting the detection of key metabolites that directly report on specific steps of cellular processes (metabolic imaging). For instance, in vivo imaging has been performed using a suitably hyperpolarized metabolic substance, and real time imaging of metabolism has been observed with pyruvate 1-13C-labeled as metabolic marker (see, for instance, Goldman K. et al, Real time metabolic imaging. PNAS 2006, 103 (30), 11270-11275) strongly suggesting the possible advantageous use of this (and other) key metabolite in the in vivo tumor diagnosis by means of hyperpolarized 13C Magnetic Resonance (MR) imaging (Albers M J. et al., Hyperpolarized 13C lactate, pyruvate, and alanine: noninvasive biomarkers for prostate cancer detection and grading; Cancer Research 2008, 68(20): 8607-15).
At the same time, the development of methods for 13C hyperpolarization has opened a new field in in vivo perfusion studies using 13C labeled hyperpolarized molecules (Mansson, S. et al, Eur. Radiol., 2006, 16, 57-67).
To this extent, the most used hyperpolarization approach relies on the application of Dynamic Nuclear Polarization (DNP) procedure that consists, essentially, in the following steps: i) preparation of a solid glassy solution of the substrate of interest with a stable organic radical; ii) bringing the solid solution to low temperature (close to 1K) into a magnet and irradiation at the frequency of the electron paramagnetic resonance (e.p.r.) transition of the organic radical for several minutes in order to transfer the electron polarization to the NMR active nuclei of the substrate molecules; iii) quick dissolution of the hyperpolarized material; iv) administration of the hyperpolarized molecules in vivo and NMR or image acquisition to report about their distribution and metabolic transformation.
Several metabolites have been polarized by means of DNP-dissolution method, among them pyruvate, acetate, fumarate, glutamate and many other metabolically interesting molecules. Commonly, the detected resonance is that of the 13C carbon atom of the carboxylate moiety, having a T1 value in the range of 20-60 s.
Overall, albeit the possibility to hyperpolarize, in principle, any substrate, the DNP-dissolution method requires sophisticated and expensive equipment. Another drawback of this technique is represented by the long polarization cycles, of about 1 hour, that are needed to reach a satisfactory nuclear polarization.
The alternative ParaHydrogen Induced Polarization (PHIP) method relies in the addition of a para-hydrogen (or parahydrogen, as herein used interchangeably) molecule to an unsaturated substrate, that allows to transform the spin order of the parahydrogen into hyperpolarization of heteronuclei.
Unlike the DNP method, the hyperpolarization procedure based on the use of parahydrogen is quite easy to handle and requires simple equipment, and offers faster preparations with hyperpolarization cycles as short as 1 min. by yielding signal-to-noise enhancements of up to 105 with only little technical effort.
A bottleneck in the use of this technique is rather represented by the limited availability of relevant unsaturated molecular precursors that are necessary for molecular addition of parahydrogen acting, as said, as a source of spin order.
Optimal substrate precursors for preparing 13C or 15N hyperpolarized molecules (to be considered as preferable for in in vivo applications by having about zero background signal and longer relaxation times T1, consenting to limit the polarization loss due to relaxation) consist of unsaturated —C═C— or —C≡C— bonds adjacent to the heteronucleus to be polarized. For 13C compounds, this represents a three carbon limitation successfully represented, for instance, by the acrylate moiety leading, after parahydrogenation, to propionate compounds finding useful application for angiographic imaging (Goldman, M. et al., C. R. Phys. 2005, 6, 575-581). However, the need of suitable substrates including a hydrogenable double or triple bonds has, in practice, strongly limited the number of hyperpolarized molecules obtainable by use of this polarization technique.
Another issue contributing to further reduce attractive unsaturated precursors is represented by the instability due to a steady intramolecular re-arrangement thereof, the so called keto-enol tautomerism, converting vinyl alcohols to the corresponding aldehydes. The PHIP parahydrogenation of an enol form stabilized by formation of a phosphate, namely phosphoenolpyruvate that forms, after parandyrogen addition, hyperpolarized phospholactate is reported by Chekmenev et al. for instance in J. Am. Chem. Soc. 2012, 134, 3957-3960.
More recently, a new way to obtain hyperpolarized molecules by means of parahydrogen, called SABRE, has been introduced that allows to achieve polarization on molecules by reversible formation of ternary adducts of the hyperpolarization substrate, parahydrogen and an organometallic complex. (Adams, R. W. et al., Science 2009. 323, 17081711). This method allows to achieve polarization on molecules without that the hydrogen addition takes place, so one of the main limits of PHIP application can be circumvented.
Furthermore, a one-step method to obtain aqueous solution of parahydrogenated molecules which relies on the parahydrognation, in organic phase, of an alkenylic or alkynylic precursor of the desired hyperpolarized alkylic or alkenylic product, followed by its quick transformation into the final molecule and extraction in an aqueous phase is disclosed by the Applicant in WO 2010/037771. By using of this method, an aqueous solution of succinic acid has been, for instance, obtained by parahydrogenation of maleic anhydride in chloroform/acetone mixture, followed by dilution with basic aqueous solution and phase transfer.
However, at the best of our knowledge, 13C-hyperpolarized molecules of diagnostic interest, such as, especially, 13C-hyperpolarized acetate and pyruvate, are currently only available with DNP hyperpolarization technique (Kohler, S. J. et al.; Magn Reson. Med. 2007, 58, 6569), while their preparation with PHIP technique is deemed highly challenging, if not unfeasible (see the aforementioned J. Am. Chem. Soc. 2012, 134, 3957-3960).
Suitable unsaturated substrates and a preparation procedure have been now identified, herein disclosed, allowing to obtain [1-13C]-hyperpolarized carboxylate containing molecules of diagnostic interest that are not directly obtainable with PHIP technique by addition of para-hydrogen to their one unsaturated direct precursor and are thus currently obtained by means of the DNP hyperpolarization technique.
In particular, an alternative PHIP-based polarization process is herein proposed that comprises using appropriate unsaturated esters, herein identified, as suitable hydrogenable substrate precursors for preparing [1-13C]-hyperpolarized carboxylate containing molecules of diagnostic interest.
According to the proposed process, a suitable unsaturated ester of the carboxylate molecule of interest is obtained and hydrogenated with molecular para-hydrogen, thereby giving to the corresponding parahydrogenated ester; a polarization transfer is then induced from added H atoms to the 13C signal of the carboxylate carbon atom by known means, for instance trough application of a field cycling, yielding the [1-13C]-hyperpolarized hydrogenated ester that is converted, by removal of the hydrogenated group, into the desired [1-13C]-hyperpolarized free carboxylate molecule, that is finally collected in an aqueous solution as such, or in the protonated form, as the corresponding carboxylic acid.
Carboxylate containing molecules of diagnostic interest according to the invention are both the carboxylate compounds finding suitable application as MR probes in Magnetic Resonance Imaging (MRI) or Magnetic Resonance Spectroscopy (MRS), typically in vascular imaging, perfusion mapping, interventional imaging or molecular imaging, and the carboxylate molecules that are key part of biologic processes and metabolic pathways such as, for instance, tricarboxylic acid (TCA) cycle (also known as citric acid cycle), glycolysis, beta-oxidation, urea cycle and ketobody metabolic pathways, that find, instead, an always increasing use as metabolic MR markers.
Optimal unsaturated esters for the use of the invention, namely as suitable hydrogenable substrate precursor of the above carboxylate containing molecule, are vinyl esters, which have the 13C carboxylate carbon atom at the desirable three bond distance from the proton added with para-hydrogenation of the vinyl group.
However, due to the impossibility to easily have suitable ethenol or ethynol alcohol because of the keto-enolic equilibrium, a convenient preparation of the appropriate vinyl ester of carboxylate molecule of interest is sometime hardly feasible, as is the case, for instance, of the pyruvate, or impractical on a scale consenting to satisfy an always increasing medical demand.
We have now unexpectedly seen that, despite this involve moving the hydrogenable unsaturation at a distance from 13C carboxylate carbon atom exceeding the optimal three bond conventionally used and recommended as necessary to have a satisfactory polarization transfer, a different family of unsaturated esters, for instance including allyl and propargyl esters, can be conveniently be prepared and used as suitable hydrogenable precursor molecules allowing to get the corresponding [1-13C]-hyperpolarized carboxylate molecules of interest, or the corresponding carboxylic acid, with good yield and satisfying polarization degree, that has proven to be substantially the same obtainable with an adjacent unsaturated carbon-carbon bond such as, for instance, that of a vinyl ester.
The hydrogenation with molecular para-hydrogen of these unsaturated esters of the carboxylate containing molecules of interest has indeed proven to allow a convenient PHIP-based preparation of the corresponding [1-13C]-hyperpolarized carboxylate molecules, or of the corresponding carboxylic acids, that can finally be collected in aqueous solutions ready for use in in-vivo MR applications.
The present invention, therefore, generally relates to a process making use of the Para-Hydrogen Induced Polarization technique for the preparation of [1-13C]-hyperpolarized carboxylate containing molecules of diagnostic interest that comprises hydrogenating with molecular para-hydrogen an unsaturated alkenyl or alkynyl ester of the concerned carboxylate molecule, used as a suitable hydrogenable substrate precursor of the carboxylate molecule of interest.
More particularly, in one embodiment the invention relates to a PHIP process for the preparation of [1-13C]-hyperpolarized carboxylate containing molecules for use in MR diagnostic applications that comprises the steps of:
a) obtaining an unsaturated alkenyl or alkynyl ester of the carboxylate containing molecule of interest and reacting the unsaturated ester with molecular para-hydrogen, to give the corresponding parahydrogenated ester;
b) inducing a polarization transfer from added polarized H to the 13C signal of the [1-13C]-carboxylate carbon atom to give the corresponding [1-13C]-hyperpolarized carboxylate ester;
c) removing the hydrogenated ester moiety and collecting an aqueous solution of the [1-13C]-hyperpolarized carboxylate containing molecule, or of the corresponding [1-13C]-hyperpolarized carboxylic acid.
In a different embodiment, the above process further comprises employing the collected aqueous solution of the [1-13C]-hyperpolarized carboxylate molecule of interest for the in vivo or in vitro, ex vivo MR assessment of biological parameters or metabolic profiles of diagnostic interest.
In a further embodiment the invention relates to the use of unsaturated alkenyl or alkynyl esters as suitable hydrogenable substrate precursor for the preparation of [1-13C]-hyperpolarized carboxylate containing molecules of diagnostic interest for MR applications by means of the PHIP technique.
In another embodiment, the invention relates to parahydrogenated alkyl or allyl esters according to the invention as intermediate compounds in a PHIP process for preparing [1-13C]-hyperpolarized carboxylate containing molecules for use as metabolic markers.
In a still additional embodiment, the invention relates to a method for the in vivo or in vitro (ex vivo) diagnostic visualization of a body organ, region, fluid or tissue or for the MR-based assessment of a biological parameter or a metabolic profile of diagnostic interest in an individual patient that comprises:
The present invention is directed to unsaturated alkenyl or alkynyl esters and a Para-Hydrogen Induced Polarization process using them as hydrogenable substrate precursor for the preparation of [1-13C]-hyperpolarized carboxylate containing molecule of diagnostic interest, especially as MR metabolic probes.
Carboxylate containing molecules according to the invention (whose [1-13C]-hyperpolarized derivatives can be conveniently prepared by use of the Para-Hydrogen Induced Polarization process proposed by the present invention) preferably include carboxylic compounds having the following general formula (I)
R—C.(O)—OH (I)
in which
With C1-C5 linear or branched alkyl chain, or C1-C5 alkyl residue, as used herein, we intend any of the C1-C5 alkyl residue, thus including methyl, ethyl, n-propyl, n-butyl, n-pentyl, isopropyl, isobutyl, sec-butyl, tert-butyl, 2-methylbutyl, where methyl, ethyl, propyl and isopropyl are preferred. With halogens (or halogen atoms, as herein used interchangeably), either alone or as part of a group (e.g. halogeno-alkyl) we refer to chlorine, bromine and fluorine, the latter being preferred.
Halogeno-alkyl groups according to the invention include, for instance perfluorinated C1-C3 alkyl residues, i.e. any C1-C3 alkyl residues wherein all the hydrogen atoms are replaced by fluorine atoms, like, for instance, —CF3 —C2F5, and —C3F7 groups, where —CF3 (i.e. trifluoromethyl) is preferred.
Examples of carbocyclic rings according to the invention for instance include either aliphatic or aromatic C6 membered rings such as, preferably, phenyl rings.
Preferably in the above formula (I) R represents a group selected from a C1-C5 alkyl residue of formula —C1-C5, a methylcarbonyl of formula CH3C(O)—, a hydroxyalkyl such as, preferably, the hydroxyethyl residue of formula CH3CH(OH)—, and an aminoalkyl residue of formula R2—CH(NHR1)— in which R1 is as defined above, and R2 is H or C1-C4 linear or branched alkyl chain such as, preferably, methyl, ethyl, propyl, isopropyl, sec-butyl, and iso-butyl, which is optionally substituted by a hydroxy (—OH) group, or a phenyl or hydroxyphenyl ring.
More preferably, R is selected from the group consisting of methyl, propyl, hydroxyethyl, methylcarbonyl and an aminoalkyl residue of formula R2—CH(NHR1)— in which R1 is H and R2 is H or C1-C4 linear or branched alkyl chain as above defined, which is optionally substituted by a hydroxy (—OH) group, or a phenyl or hydroxylphenyl ring, such as, most preferably, isopropyl, iso-butyl, sec-butyl, 1-hydroxyethyl, hydroxylmethyl and p-hydroxybenzyl.
In one preferred embodiment, in the above formula (I) R is a methyl residue (—CH3), and the carboxylate containing molecule of interest according to the invention is the acetate.
In another preferred embodiment, in the above formula (I) R is the hydroxyethyl residue of formula CH3CH(OH)— and the carboxylate containing molecule of interest according to the invention is the lactate.
In a further preferred embodiment, in the above formula (I) R is an amino alkyl group of formula R2—CH(NHR1)— in which R1 is H and R2 is H, or a group selected from isopropyl, iso-butyl, sec-butyl, hydroxymethyl, 1-hydroxyethyl and p-hydroxybenzyl, and the carboxylate containing molecule of interest according to the invention is a natural amino acid.
In an especially preferred embodiment of the invention, in the above formula (I) R is a methylcarbonyl group of formula CH3C(O)— and the carboxylate containing molecule of interest according to the invention is the pyruvate.
Carboxylate containing molecule according to the invention further comprise, as formerly said, the physiologically acceptable salts of the compounds of the above formula (I).
With pharmaceutically acceptable salt, as used herein, we refer to derivatives of the carboxylate compounds of the above formula (I) in which the carboxylic group not yet internally neutralized is in the form of a non-toxic, stable salt which do not destroy or affect the activity of the hyperpolarized compound.
Suitable examples of the said salts typically include alkali or organic salts of the carboxylic acidic residue of formula (I).
To this extent, preferred cations of inorganic bases which can be suitably used to salify the compounds of the invention comprise ions of alkali or alkaline-earth metals such as potassium, sodium, calcium or magnesium. Preferred cations of organic bases comprise, inter alia, those of primary, secondary and tertiary amines such as ethanolamine, diethanolamine, morpholine, glucamine, N-methylglucamine, N,N-dimethylglucamine. Preferred cations of amino acids comprise, for example, those of aspartic and glutamic acids.
Particularly preferred are sodium and potassium salts.
The carboxylate compounds set forth by the above formula (I) do not include a suitable unsaturated carbon-carbon bond in the R residue, thus they cannot be hyperpolarized by suitable addition of molecular para-hydrogen to an existing unsaturation bond and polarization transfer to the 13C carbon atom of the carboxylate group.
The solution proposed by the present invention comprises obtaining unsaturated alkenyl or alkynyl esters, and preferably, C2-C4 linear or branched alkenyl or alkynyl esters of the above carboxylate compounds of formula (I) and using the obtained unsaturated esters as convenient hydrogenable substrate precursors of the desired [1-13C]-hyperpolarized carboxylate molecules.
Within the scopes of the present invention, the terms “substrate precursor” or “hydrogenable precursor” or “unsaturated precursor” or, simply, “precursor”, used herein interchangeably, refer to an unsaturated molecule including one unsaturated bond, e.g. a double or a triple carbon-carbon bond, yielding the desired hyperpolarized product by hydrogenation of the said unsaturated bond with molecular para-hydrogen, transfer of the polarization to a non-proton nucleus, and conversion of the obtained hydrogenated compound in the desired hyperpolarized product.
Example of hyperpolarizable non-proton nucleus (or heteronucleus) typically include a 19F, 13C, 15N or 29Si. According to present invention, unless otherwise provided, with the term hyperpolarizable heteronucleus we refer to a 13C, and, more particularly, we refer to the 13C-carboxylic carbon atom (or 1-13C-carbon atom) of the carboxylate molecule of interest.
Substrate precursors for the use of the present invention are unsaturated alkenyl or alkynyl esters of the carboxylate molecule of interest, namely esters that include one unsaturated bond, e.g. a double or a triple carbon-carbon bond in the hydrocarbon R′ chain of the —O—R′ ester moiety, and are easily hydrogenable.
An object of the present invention thus relates to the use of an unsaturated alkenyl or alkynyl ester of a carboxylate containing molecule of diagnostic interest as hydrogenable substrate precursor for the preparation of the corresponding [1-13C]-hyperpolarized carboxylate containing molecule by use of the PHIP technique.
More particularly, the present invention relates to a PHIP process for the preparation of [1-13C]-hyperpolarized carboxylate containing molecules of diagnostic interest which comprises hydrogenating with molecular para-hydrogen unsaturated alkenyl or alkynyl ester of the concerned carboxylate molecule.
To this extent, unsaturated alkenyl or alkynyl esters according to the invention, namely for use as hydrogenable substrate precursor for the preparation of [1-13C]-hyperpolarized carboxylate compound with the PHIP technique, preferably include C2-C4 linear or branched alkenyl or alkynyl esters of the carboxylate containing molecule of interest that, more preferably, are selected from vinyl, allyl or propargyl esters of the said concerned carboxylate molecule.
According to a preferred embodiment, the unsaturated esters according to the invention are 13C enriched, or 13C labeled, as herein used interchangeably. The term “enriched” or, alternatively “labeled” means that the concentration of the non-zero spin nucleus in the compound is above the typical value of natural abundance of said nuclei, preferably above at least 10% of natural abundance, more preferably above at least 25%, and even more preferably above at least 75% of its natural abundance and most preferably above at least 90% of its natural abundance. According to the present invention, the enrichment is concentrated on the carboxylic 1-C carbon atom of the ester that becomes 13C-enriched (or [1-13C]-enriched, as herein used interchangeably). Said non-zero 13C nucleus confers to the obtained carboxylate containing molecule (or carboxylate product, or, simply, product, as used herein interchangeably) a T1 relaxation time of at least 5 seconds (indicated with s), preferably of at least 10 s, preferably of at least 20 s, preferably of at least 30 s, and even more preferably of at least 40 s, measured in a solution subjected to a magnetic fields of from about 0.5 mT to about 20 T (Tesla). The appropriate 13C enrichment may be natural, when the carboxylate ester is naturally 1-13C enriched, or it may include the selective enrichment (or 13C labeling, as herein used alternatively) of the carboxylic 1-C carbon atom of the molecule. To this extent, commercially available enriched precursors can be suitably employed or, in case, the enrichment of choice can be achieved by chemical synthesis, or biological labeling, according to well-known prior art teachings.
Unsaturated esters for the use of the instant invention as substrate precursor should be highly polarisable. In particular, preferred esters are polarisable to a degree corresponding to at least 5%, preferably at least 10% and, more preferably of at least 20% or even higher, and are capable to maintain a 13C net magnetization in the carboxylate product within the above limits after removal of the hydrogenated moiety and isolation of the free hyperopolarized carboxylate molecule of diagnostic interest.
To this extent, after hydrogenation with molecular para-hydrogen and transfer of the polarization to the carboxylic 13C-carbon atom, preferred unsaturated ester according to the invention should allow the easy removal of the hydrogenated moiety in an aqueous medium, typically by hydrolysis, thus enabling a quick isolation of an aqueous solution of the free [1-13C]-hyperpolarized carboxylate molecule, ready for use in in vivo MR applications.
In general terms, the unsaturated esters for the use of the invention can be, preferably, represented by the following general formula (II)
R—C.(O)—O—R′ (II)
in which:
C. denotes the naturally 13C enriched or, optionally, 13C labeled carboxylate carbon atom undergoing 13C hyperpolarization,
R is as above defined for the formula (I), and
R′ is a C2-C4 linear or branched hydrocarbon chain comprising a double or triple carbon-carbon bond.
Preferably, in the above formula (II) R′ is an alkenyl or alkynyl residue selected from vinyl (of formula —CH═CH2), allyl (of formula —CH2—CH═CH2) and propargyl (of formula —CH2—C≡CH).
More preferably, R′ is a vinyl or a propargyl residue.
In one preferred embodiment, the invention refers to unsaturated esters of the above formula (II) in which R′ is a vinyl residue.
In another preferred embodiment, the invention refers to unsaturated esters of formula (II) in which R′ is a propargyl residue.
An additional object of the invention relates to a process that comprises hydrogenating with molecular para-hydrogen an unsaturated alkenyl or alkynyl ester, preferably having the above formula (II), for preparing [1-13C]-hyperpolarized carboxylate containing molecules of diagnostic interest, especially as MR metabolic probes, by use of the Para-Hydrogen Induced Polarization technique.
More particularly, in one preferred embodiment the invention relates to a process for the preparation of [1-13C]-hyperpolarized carboxylate containing molecules of formula (I) that comprises the steps of:
a) obtaining an unsaturated alkenyl or alkynyl ester of the carboxylate containing molecule of interest and reacting the unsaturated ester with molecular para-hydrogen, to give the corresponding parahydrogenated ester;
b) inducing a polarization transfer from added para-hydrogen to the 13C signal of the [1-13C]-carboxylate carbon atom to give the corresponding [1-13C]-hyperpolarized ester;
c) removing the hydrogenated ester moiety and collecting an aqueous solution of the [1-13C]-hyperpolarized carboxylate containing molecule, or of the corresponding [1-13C]-hyperpolarized carboxylic acid.
Preferably, the unsaturated ester obtained at the step a) of the process is an alkenyl or alkynyl ester of the above formula (II).
To this extent, suitable unsaturated esters of the above formula (II) can be commercially available, as is the case, for instance, of the vinyl acetate, or may be conveniently obtained by using conventional preparation procedures, easily available to those skilled in synthetic organic chemistry techniques. The preparation of non-limiting examples of representative unsaturated esters according to the invention is moreover provided in the experimental section which follows.
The hydrogenation of the unsaturated ester according to the step a) of the process of the invention is performed by use of a PHIP technique, in the presence of a suitable hydrogenation catalyst. Typically, the latter is used in catalytic amounts known to a skilled person, for instance in a substrate/catalyst ratio ranging from 10:1 to 5:1.
A further embodiment of the invention therefore relates to parahydrogenated alkyl or allyl esters, e.g. obtained by hydrogenating with molecular para-hydrogen unsaturated esters according to the invention, preferably of the above formula (II), finding application as intermediate compounds in a PHIP process for preparing hyperpolarized molecules, e.g. for preparing [1-13C]-hyperpolarized carboxylate containing molecules for use as MR metabolic markers.
More particularly, in a further embodiment the present invention relates to a para-hydrogenated ester of formula (III)
R—C.(O)—O—R″ (III)
in which C. and R are as above defined, and R″ is a parahydrogenated alkyl or allyl residue such as, preferably, a parahydrogenated ethyl, propyl or allyl and, most preferably, ethyl or allyl, e.g. obtained by hydrogenation with molecular para-hydrogen of an unsaturated ester of the above formula (II) in which R′ represents, respectively, a vinyl, an allyl or a propargyl and, most preferably, a vinyl or a propargyl residue, as well as the use thereof in a PHIP process, e.g. as intermediate compound for the preparation of [1-13C]-hyperpolarized carboxylate containing molecules.
In an additional embodiment the invention relates to a process for the preparation of hyperpolarized molecules by use of the Para Hydrogen Induced Polarization technique that comprises obtaining a para-hydrogenated ester of formula (III) as an intermediate compound.
Suitable examples of the para-hydrogenated intermediates of formula (III) according to the invention for instance include the ethyl acetate obtained by addition of molecular para-hydrogen to a the vinyl acetate precursor, which 13C-NMR characterization is provided in
According to the process of the present invention, the hydrogenation of the unsaturated ester with molecular para-hydrogen can conveniently be carried out either in an aqueous medium, or in an organic solvent, or suitable mixtures of organic solvents.
In one embodiment of the invention, the steps a) of the process including reacting the unsaturated ester with molecular para-hydrogen is carried out in an aqueous solvent and in the presence of a water soluble hydrogenation catalyst, according to hydrogenation procedures known in the art.
To this extent, with the expression “aqueous solvent” or “aqueous medium”, as used herein interchangeably, we refer to water and, preferably, to sterile water, which can optionally be properly buffered, for instance at an around neutral pH value, for instance comprised from 6 to 8, and, preferably, of about 7, by use of appropriate buffers, such as, for instance, phosphate buffer (H3PO4/H2PO4).
Examples of catalysts suitable for the use in aqueous solvents include rhodium(I) complexes of formula [Rh(diphosphine)diene)]+[anion]−, where the diphosphine is a chelating phosphine preferably selected from (1,4-Bis(R1R2)ethane), (1,4-Bis(R1R2)butane), where R1 and R2, equal or different the each other, comprise sulfonated groups such as, for instance, DPPETS (1,2-bis[bis(m-sodiosulphonatophenyl)phosphino]ethane)(1,2-Bis(diphenylphosphino)ethane), DPPBTS (tetrasulfonated 1,4-bis(diphenylphosphino)butane, in which R1═R2), DAPBTS (tetrasulfonated bis(dianisylphpsphino)butane), chiral sulfonated diphosphines such as, for instance, sulfonated CHIRAPHOS (2,3-bis[bis(m-sodiosulphonatophenyl)phosphino]butane), and sulfonated BINAP (2,2-bis[bis(m-sodiosulphonatophenyl)phosphino]-1,1-binaftile). The diene is preferably selected from 1,5-cyclooctadiene and norbornadiene, and the anion can be any anion, but, preferably, tetrafluoroborate or trifluoromethyl solfonate.
Preferred are hydrogenation catalysts in which the phosphine group is diphenylphosphinobutane, and the [Rh(NBD)phos][BF4] (where NBD is norbornadiene, and phos is 1,4-bis[(phenyl-3-propane sulfonate)phosphine]butane disodium salt) is particularly preferred.
According to one practical implementation, the hydrogenation reaction of the unsaturated substrate according to the invention is suitably carried out by use of a hyperpolarizer where hydrogen is introduced at high pressure, typically higher than 6 and preferably of at least 8 bar, into a reactor chamber causing the nebulization of the solution containing the substrate and the catalyst, and which allows to optimize the hydrogen solubility. The hydrogenation reaction is preferably carried out at a temperature comprised from 30° C. to 90° C. and, more preferably, from 60° C. to 90° C.
It is clear to those skilled in the art that when the hydrogenation reaction is carried out in an aqueous medium, as above disclosed, either the hydrogenation catalyst and the unsaturated substrate molecule should be soluble in this medium.
To this extent, a little amount of a short-chain alcohol, such as, for instance, methanol or ethanol or, alternatively of any water soluble organic solvent such as, preferably, acetone, can be added to the aqueous medium, in an amount at least higher than 10%, preferably ranging from 10% to 30% and, more preferably, from 10% to 20% of the total solvent amount, consenting to increase both the catalyst efficacy and the substrate and hydrogen gas solubility in the aqueous medium.
Alternatively, a short-chain alcohol such as, preferably, methanol or ethanol and, more preferably, methanol, can be suitably used as hydrogenation reaction solvent. In this case, preferred hydrogenation catalyst is [Rh(COD)dppb][BF4], where COD is cyclo-1,5-octadiene and dppb is 1,4-bis(diphenylphosphino)butane).
Upon hydrogenation of the unsaturated ester with para-hydrogen, the polarization is transferred from the added polarized H to the 13C signal of the [1-13C]-carboxylate carbon atom via scalar coupling (or nuclear Overhauser effect) by use of the know means.
To this extent, in order to profitably use a parahydrogenated compound as an effective 13C MRI contrast agent, it is necessary that the “anti-phase” signal of the hyperpolarized carbon atom, obtained through polarization transfer from the added parahydrogen, is totally converted in an “in-phase” signal, useful for imaging acquisition. This step can be performed by using an appropriate pulse sequence as taught, for instance, by Goldman M. et al, in the above cited reference (C. R. Phisique 2005, 6, 575), or by applying an appropriate field cycling procedure to the parahydrogenated product. This last includes rapidly introducing (non-adiabatically) the hydrogenated sample into a magnetic screen (field intensity=0.1 μT), and then slowly removing (adiabatically) the screen to bring the sample to field values corresponding to the Earth's magnetic field (50 μT) (in this respect see, for instance, C. R. Phisique 2004, 5, 315).
According to a preferred embodiment, the step b) of the process of the invention is carried out by application of an appropriate field cycling procedure to the para-hydrogenated ester obtained at the step a) of the process, consenting to promote the polarization transfer from the proton nuclei (deriving from the parahydrogen addition to the unsaturated ester) to the non-proton nucleus of interest, namely the [1-13C]-carbon atom of the carboxylate molecule to give the corresponding [1-13C]-hyperpolarized carboxylate molecule.
It is worth nothing that, in general terms, the polarization transfer from para-hydrogen to the heteronucleous of interest is mainly driven by scalar coupling (or j coupling), and a heteroatom adjacent to the unsaturation typically acts as recipient of the polarization transfer from the scalarly coupled para-hydrogen protons (as shown, for instance, in
From all the foregoing, a skilled person in the art would have never expected that one could observe a satisfactory heteronuclear polarization, useful for MR diagnostic applications and metabolic MR assessments, by polarization transfer from parahydrogen protons placed at a distance of more than two or three bonds from the 13C carbon atom of interest. Even less one would have expected to could observe a heteronuclear 13C polarization substantially equivalent or comparable to that obtained with a heteronucleus placed at the (above identified) optimal distance with 13C carbon atoms rather placed at a distance of 4-5 bonds from the added para-hydrogen protons.
Instead, we have unexpectedly observed that, after application of magnetic field cycle, a substantially identical hyperpolarization of the 13C signal of the [1-13C]-carboxylate carbon atom can be achieved by polarization transfer from both the polarized H proton nuclei of the parahydrogenated vinyl ester (having the recommended 3 bond distance to the involved 13C heteronucleous) and the polarized H proton nuclei of the corresponding parahydrogenated propargyl ester, having an increased (4-5 bonds) distance to the heteronucleous of interest, as shown, for instance, in
On the contrary, as shown in
These unexpected results make it possible to prepare esters suitably hyperpolarized to the carboxylic [1-13C]-carbon atom and, in turn, to obtain suitably [1-13C]-hyperpolarized carboxylate containing molecules of diagnostic interest from unsaturated allyl or propargyl precursor thereof, thereby allowing to obtain [1-13C]-hyperpolarized carboxylate compounds endowed with a satisfactory degree of polarization by use of the PHIP technique even in the absence of a direct unsaturated precursor thereof, as well as of an appropriate vinyl ester.
Rightly after its preparation, the [1-13C]-hyperpolarized ester for instance obtained at the step b) of the process of the invention is quantitatively converted into the desired [1-13C]-hyperpolarized carboxylate compound, or in the corresponding [1-13C]-hyperpolarized carboxylic acid, that is then collected in an aqueous solution ready for use in in vivo applications. According to a preferred embodiment of the invention, the said quantitative conversion is performed by hydrolytic removal of the hydrogenated ester moiety, yielding the free carboxylate of interest, or the corresponding carboxylic acid.
To this extent, the expression “quantitative conversion” is herein used to indicate a chemical transformation (preferably a hydrolysis) in the amount of 20% or more, preferably 50% or more, more preferably 75% or more and even more preferably of at least 90%, particularly preferred being a transformation of at least 95% of the ester precursor into the corresponding free carboxylate.
The term “hydrolysis”, as used herein, refers to a chemical reaction in which the water reacts with a starting compound to produce one or more resulting compound(s); it typically involves the splitting of a bond (the ester bond) on the starting compound and the optional addition of a hydrogen cation and/or of a hydroxide anion to the structure of the starting compound, to obtain the resulting compound(s). Generally speaking, the hydrolysis reaction can be carried out under acidic (pH<7), basic (pH>7) or even neutral conditions (pH=7), whereas basic conditions, for instance corresponding to a pH solution preferably comprised from 7 to 14, more preferably form 8 to 14 and, most preferably, from 10 to 14 are to be considered as particularly preferred for the process of the present invention, as will be described herein below in more details.
In line with all the above, the step c) of the process of the invention comprises hydrolyzing the [1-13C]-hyperpolarized ester obtained at the step b) of the process to the corresponding water soluble [1-13C]-hyperpolarized carboxylate containing compound, that is then collected in an aqueous solution.
More particularly, according to one preferred implementation, the step c) of the process of the process of the invention is carried out by adding the [1-13C]-hyperpolarized ester obtained at the step b) of the process in an aqueous solution with a suitable amount of a base, e.g. NaOH, or NaHCO3, or Na2CO3, as well as organic or inorganic compounds having basic aqueous reaction (e.g. trimethylol aminomethane, also known as tromethamine, or trisodium phosphate) for instance as schematized in schemes a) and b) of
Particularly preferred for the scope of the instant invention is the use of aqueous NaOH.
For instance, an aqueous solution of the hyperpolarized ester obtained at the step b) of the process with a 10-100 mM concentration is hydrolyzed in the presence of NaOH 0.1-1 M, that is added to the aqueous solution at a temperature ranging from about 20° to 100° C., preferably from 40° to 80° C. and most preferably from 60° to 80°, thereby leading to an aqueous solution of the [1-13C]-hyperpolarized carboxylate compound.
To this extent, it must be considered that, for in vivo MR applications, the biocompatibility of the obtained aqueous solutions of hyperpolarized products is required. Therefore, after steps a) to c), the above process according to the invention comprising carrying out the 13C polarization of the precursor ester molecule in an aqueous medium as above described preferably includes an additional step d) comprising removing hydrogenation catalyst and optional organic co-solvent(s) from the aqueous solution of the [1-13C]-hyperpolarized carboxylate compound obtained at step c), thereby obtaining an aqueous solution of [1-13C]-hyperpolarized carboxylate containing molecule of interest suitable for use in in vivo applications.
This last task can, for instance, be achieved by quick evaporation of the aqueous solution of the hyperpolarized product, for instance by spraying the solution into a chamber or flask connected to a vacuum pump. The removal of the potentially toxic Rh(I) complex may then be conveniently performed, e.g. by elution of the aqueous solution (of the hyperpolarized molecule) resulting from removal of any optional organic solvent or co-solvent on a micro-column containing less than 1 ml of a cationic exchange resin, retaining the positively charged hydrogenation catalyst.
To overcome all the above purification steps, according to a particularly preferred embodiment, the steps a) of the process of the invention, including reacting the unsaturated ester with molecular para-hydrogen, is carried out in an organic solvent, or a suitable mixture of organic solvents, and in the presence of a hydrogenation catalyst soluble in an organic medium and insoluble in an aqueous solvent.
Organic solvents suitable for the purpose of the invention are immiscible with water and, preferably, comprise organochlorinated solvents such as, for example, chloroform, dichloromethane, carbon tetrachloride, ethers such as, for instance, diethylether, diisopropylether and butylether and aliphatic hydrocarbons such as, for instance, pentane, hexane, heptane, octane and cyclohexane, ethyl acetate and so on.
Among them, preferred are chlorinated solvents, wherein particularly preferred are chloroform and dichloromethane as well as suitable mixtures thereof or, optionally, mixtures of the above solvents with a minimum amount of a short-chain alcohol such as, typically, ethanol or, preferably, methanol, or, alternatively of acetone. To this extent, with minimum amount as used herein we intend an amount which is less than 30%, preferably ranging from 10% to 30%, and, more preferably, from 10 to 20% of the total solvent amount, leading to undetectable residues in the aqueous solutions of the hyperpolarized carboxylate containing molecules finally recovered. Optional traces, e.g. obtained by using higher amounts of water-miscible solvents, can on the other side, be suitably removed from collected aqueous solution of the desire hyperpolarized molecule, for instance by its rapid evaporation or spry-drying, as formerly discussed.
Examples of catalysts suitable for the use of the present invention include rhodium complexes of formula [Rh(diphosphine)diene)]+[anion]−, where the diphosphine is preferably selected from DPPB (1,4-Bis(diphenylphosphino)butane), DPPE (1,2-Bis(diphenylphosphino)ethane) and derivatives thereof including, for instance, the chiral phosphines such as DINAP (2,2′-Bis(diphenylphosphino)-1,1′-binaftyl), CHIRAPHOS (2,3-diphenylphosphinobutane), DIOP (1,4-Bis(diphenylphosphino)-1,4-bisdeoxy-2,3-O-isopropyliden-L-treitol), and DIPAMP (1,2-Bis[(2-methoxyphenyl)(phenilphosphino)]ethane); the diene is preferably selected from 1,5-cyclooctadiene and norbornadiene, and the anion can be any anion, but, preferably, tetrafluoroborate or trifluoromethyl solfonate.
Among them, preferred are catalysts in which the phosphine group is diphenylphosphinobutane, while the [Bis(diphenylphosphinobutane)(1,5-cyclooctadiene)]Rh(I) is particularly preferred.
In practical terms, the hydrogenation of the unsaturated substrate is, for instance, carried out by spraying the organic solution comprising the substrate and the catalyst into a reaction chamber previously pressurized with para-H2 at a pressure preferably comprised from 6 to 10, more preferably from 8 to 10 and most preferably with about 10 atm of para-hydrogen. The hydrogenation reaction is preferably carried out at a temperature comprised from 40° to 90° C. and, more preferably, from 70° to 90°.
Right upon hydrogenating of the unsaturated ester with para-hydrogen, the polarization is transferred from the added polarized H to the 13C signal of the [1-13C]-carboxylate carbon atom.
To this extent, according to a preferred implementation, the polarization transfer according to step b) of the process of the invention is carried out by application of an appropriate field cycling procedure to the parahydrogenated ester, for instance obtained at the step a) of the process, as above discussed, promoting the desired polarization transfer from added proton nuclei to the [1-13C]-carbon atom of the carboxylate molecule (i.e. the hydrogenated ester) thus giving an organic solution of the corresponding [1-13C]-hyperpolarized ester.
The hydrolytic removal of the hydrogenated ester moiety is then carried out according to the step c) of the proposed process, yielding the desired water soluble [1-13C]-hyperpolarized carboxylate compound, or the corresponding [1-13C]-hyperpolarized carboxylic acid, that is then collected as an aqueous solution, ready for use in in vivo applications.
According to a preferred embodiment, the step c) of the process of the invention is carried out by simply diluting the organic solution of the hyperpolarized ester preferably obtained, as said, at the step b) of the process with an appropriate aqueous solution promoting the hydrolysis of the [1-13C]-hyperpolarized ester to the corresponding water soluble [1-13C]-hyperpolarized carboxylate compound, or corresponding carboxylic acid, for instance as schematically shown in
It stems from the foregoing that, according to a particularly preferred embodiment, the process of the invention comprises obtaining an insoluble or scarcely water soluble [1-13C]-hyperpolarized ester in an organic medium, e.g. from an unsaturated ester of formula (II) soluble in an organic solvent but insoluble or scarcely water soluble, for instance as above described; quantitatively converting it into the corresponding water soluble [1-13C]-hyperpolarized carboxylate compound by hydrolytic removal of the hydrogenated ester moiety carried out by dilution of the organic solution (of the hyperpolarized ester) with a suitable aqueous solution, and then collecting the obtained [1-13C]-hyperpolarized carboxylate compound in the aqueous phase, by phase transfer extraction, as an impurity-free aqueous solution ready for use in in vivo application.
According to the present invention, and unless otherwise indicated, the expressions “poorly water soluble” or “scarcely water soluble”, used herein interchangeably with reference to an unsaturated ester of formula (II) or a [1-13C]-hyperpolarized ester according to the invention, refer to a compound that has a minimal solubility in water, preferably less than 20%, more preferably, less than 5% and, even most preferably, less than 1% of the total compound amount.
On the other side, and unless otherwise indicated, the terms “aqueous solution” or “suitable aqueous solution” or “appropriate aqueous solution”, herein used interchangeably, refer to a sterile water or saline solution, optionally properly buffered, in any case physiologically tolerable and suitable for use in in vivo diagnostic applications, or, moreover, an aqueous solution as defined above, further including a suitable amount of a properly selected reagent capable of promoting the rapid and selective conversion of the hydrogenated ester into the corresponding, water soluble, [1-13C]-hyperpolarized carboxylate compound and to generate, as a result, a physiologically acceptable aqueous solution of the same, suitable for use as such in in vivo diagnostic applications, without requiring further purification.
Suitable examples of aqueous solution according to the instant invention capable of promoting the hydrolysis of the parahydrogenated substrate to the corresponding carboxylate compound comprise a minimum amount of a base, e.g. NaOH or NaHCO3, or Na2CO3, as well as organic or inorganic compounds with basic aqueous reaction (e.g. trimethylol aminomethane, also known as tromethamine, or trisodium phosphate) or the corresponding deuterated molecules. Particularly preferred for the scope of the instant invention are water solutions of NaOH or of the corresponding deuterated molecule.
However, it is clear from all the foregoing that, when the reagent used for promoting the ester hydrolysis according to the step c) of the process of the invention is not itself physiologically acceptable, such as the case of the NaOH, its quantity in the added aqueous solution must be precisely determined, based on the stoichiometry of the reaction itself, so as to be completely used in the conversion reaction of the parahydrogenated ester to the water soluble and physiologically compatible (at physiological pH condition) free carboxylate, or the exceeding amount has to be suitably neutralized (for instance by addition of a suitable amount of an acid generating with the base a physiologically acceptable salt) so as to result in a physiologically acceptable aqueous solution of the desired carboxylate compound, which is ready for use as such in in vivo applications, without requiring any further purification and/or subsequent formulation.
An aqueous solution of the [1-13C]-hyperpolarized carboxylate containing molecule (or carboxylic acid) is, thus, directly collected from step c) of the above preferred process according to the invention, which is impurity-free and usable as such in the in vivo MRI diagnostic imaging, without need of further purification.
In this respect, no detectable traces of impurities or organic solvents have been observed in the aqueous solutions of [1-13C]-hyperpolarized carboxylate containing molecules collected after hydrolysis from experimental tests in which the hyperpolarized ester in obtained in an organic solvent, though comprising methanol or acetone as organic co-solvent. Indeed, no residues of these co-solvents, at least at detectable levels, are found in aqueous solutions obtained from tests of Example 3 (see
Hence, according to an especially preferred embodiment the instant invention relates to a Para-Hydrogen Induced Polarization process for the preparation of [1-13C]-hyperpolarized carboxylate containing molecules of diagnostic interest that comprises:
Interestingly, the [1-13C]-hyperpolarized carboxylate compound obtained according to the process of the invention as an aqueous solution ready for use as such in in vivo applications, has at least 5% of polarization consenting to provide enough sensitivity in in vivo imaging. Preferably, the polarization obtained is at least 10% and, more preferably at least 15%. On the other side, the impurity-free aqueous solution of a hyperpolarized molecule obtained according to the invention are stable for a clinically acceptable period of time; in particular preferably at least 10% of this polarization is maintained at the time of the injection, which is commonly performed rightly after the preparation of the hyperpolarized carboxylate, more preferably at least about 30% polarization is maintained, most preferably at least of about 80% polarization is maintained.
To this extent, it will be clear that the polarization method herein proposed should be carried out within the frame of time in which the hyperpolarised carboxylate molecule remains significantly polarised, shortly after being subjected to the chemical conversion (e.g. hydrolysis) of the precursor. Therefore, the administration of such active substrate and the subsequent MR measurement are preferably effected as rapid as feasible. This means that the sample, either human or non-human animal body, should be available close to the area in which the polarisation takes place.
Aqueous solutions according to the instant invention preferably include the hyperpolarized molecule of interest in a concentration ranging between 0.002 and 1.0 M and preferably between 0.01 and 0.5 M.
The impurity-free aqueous solution of a hyperpolarized molecule obtained, for instance, according to the step d) or, more preferably, as above said, directly collected from the step c) of the process, according to a particularly preferred embodiment of the invention, finds advantageous use as such, (i.e. without further purification and/or subsequent formulation) in in vitro, ex vivo and, especially, in vivo MR diagnostic imaging of a human or animal body organ, fluid, region or tissue, as well as for the diagnostic assessment of physiological parameters of diagnostic interest in an individual, human or animal, patient.
Even preferably, they may find advantageous use in the emerging field concerning the evaluation of metabolic profiles of diagnostic interest in an individual patient by use of MR imaging techniques. In particular, the assessment of the metabolic conversion of a concerned hyperpolarized carboxylate may allow to provide an evaluation of the metabolic processes in an individual patient and/or information on metabolic state of a (healthy or pathological) patient's tissue or organ.
Accordingly, in an additional embodiment, the instant invention relates to a MR contrast agent that is characterized in that it comprises, or, preferably, consists of the impurity-free aqueous solution of the [1-13C]-hyperpolarized carboxylate compound collected according to step d) or, more preferably, directly collected from step c) of the process, according to a particularly preferred embodiment of the invention.
In a particularly preferred embodiment, the said [1-13C]-hyperpolarized carboxylate compound is the [1-13C]-hyperpolarized lactate.
In an alternative, equally preferred, embodiment the said [1-13C]-hyperpolarized carboxylate compound is the is the [1-13C]-hyperpolarized acetate.
In an especially preferred embodiment, the [1-13C]-hyperpolarized carboxylate compound according to the invention is the [1-13C]-hyperpolarized pyruvate.
The invention moreover relates to a Para-Hydrogen Induced Polarization process that in addition to process steps from a) to c) or from a) to d), allowing to get an impurity-free aqueous solution of the [1-13C]-hyperpolarized carboxylate molecule of interest as above described, further comprises employing the collected aqueous solution for the MR diagnostic imaging of an individual patient or for the in vivo or in vitro, ex vivo, MR assessment of biological parameters or metabolic profiles of diagnostic interest.
More particularly, according to a further embodiment, the invention relates to a Para-Hydrogen Induced Polarization process that comprises:
To this extent, the steps of the above process including exposing the administered patient or ex vivo sample to an appropriate exciting radiation, recording the signal intensity generated by the excited nucleus, and obtaining an image of the individual patient body organ, region or tissue, or suitable estimates of the biological parameter or metabolic profile of interest from recorded signals intensity values can be suitably carried out according to conventional techniques and operative procedures well known to those skilled in the relevant art.
The invention moreover relates to a method for the in vivo diagnostic MR imaging of a body organ, region, fluid or tissue or for the in vivo or in vitro, ex vivo, MR assessment of biological parameters or metabolic profiles of diagnostic interest in an individual patient that comprises:
The above method may alternatively comprises exposing to a radiation frequency exciting the 13C-hyperpolarized carboxylate molecule a patient pre-treated with a proper amount of aqueous solution of the a [1-13C]-hyperpolarized carboxylate molecule, and the recording the signal intensity generated by the excited 13C-nucleus or, still alternatively, it can includes obtaining signal intensity values (and, in turn, estimates of biological parameters or metabolic profiles of diagnostic interest) from a collection of MRI signals acquired, at the appropriate time, by an individual patient properly treated with an effective amount of aqueous solution of the a [1-13C]-hyperpolarized carboxylate molecule and suitably exposed to a radiation frequency allowing to excite the hyperpolarized 13C-signal of the carboxylate molecule, and then digitally stored in the tomograph's console memory, or in a local or remote digital data storage device.
In this respect, unless otherwise indicated, with “individual patient” or “patient” as used herein we refer to a human or animal patient, and, preferably a human being undergoing MR diagnostic assessment.
On the other side, with “effective amount” or “suitable amount”, as herein used interchangeably, we refer to any amount of the aqueous solution of the hyperpolaryzed molecule collected according to the process of the invention and, preferably, directly collected from step c) of the preferred process implementation that is sufficient to fulfil its intended diagnostic purpose(s): i.e., for example, to acquire the signal generated by the excited nucleus of the administered carboxylate molecule and/or of any suitable metabolite or catabolite thereof.
To this end, the impurity-free aqueous solution of the[1-13C]-hyperpolarized carboxylate (or carboxylic acid) obtained by use of the method of the present invention can be administered as such into the vascular system or directly into an organ or muscle tissue, or by subdermal or subcutaneous route, as the case may be. Then, according to the present method, the sample is exposed to a uniform magnetic field (also known as “primary magnetic field”) with radiation of a frequency selected to excite nuclear spin transitions in the said hyperpolarised 13C-carboxylic signal. The hyperpolarization of the 13C-signal of the carboxylic ester and, consequently, of the corresponding carboxylate molecule, results in an increasing in the population difference between the excited and ground nuclear spin states of those nuclei which are responsible for the magnetic resonance signals. Since MR signal intensity is proportional to this population difference, the final detected MR signals result in larger amplitude signals. The amplitude of the induced MR signals is also dependent upon several other factors, such as the strength of the magnetic field, the temperature of the sample, the isotopic nature and chemical environment of the imaging nuclei and the like.
The present invention will be further illustrated by the following examples that are intended to be illustrative and are in no way limiting the scope of the invention.
Experimental Part
All the 13C-NMR spectra are acquired into a 600 MHz Bruker spectrometer, by operating at 14.1 T, and 298 K.
All the chemicals and reagents used are commercially available or can be prepared according to well-known methods of the art.
The Parahydrogenation of Vinyl-Acetate
The parahydrogenation of vinyl-acetate (purchased by Sigma-Aldrich; reference code V1505) has been carried out in water using the water soluble catalyst [Rh(NBD)phos][BF4] (NBD=norbornadiene, phos=1,4-bis[(phenyl-3-propane sulfonate)phosphine]butane disodium salt) (2.5 mM deuterated water solution) (this catalyst can be prepared e.g. according to the procedure reported in Magn. Reson. Mat. Phys. Biol. Med. 22, 123-34 (2009). The parandyrogenation reaction has been carried out into a NMR tube equipped with Young valve charged with 0.3 ml of catalyst, 0.03 mmol of substrate (3 μl, final concentration about 100 mM), 0.05 ml of methanol and 8 bar of parahydrogen (enriched at 77K, 50% enrichment).
Preparation of the [1-13C]-Hyperpolarized Ethyl Ester
After warming the solution at 90° C., the NMR tube was vigorously shaken for 10″, then the 13C spectrum has been immediately acquired. In
Preparation of the [1-13C]-Hyperpolarized Acetate
To get the corresponding [1-13C]-hyperpolarized acetate, the para-hydrogenation reaction has repeated as above described with a second amount of vinyl-acetate. The obtained [1-13C]-hyperpolarized ester is then hydrolysed by addition of 0.05 ml of NaOD 6M immediately after field cycling procedure and a 13C-NMR spectrum is then acquired.
Synthesis of Propargyl Acetate
Acetic acid (2.00 g, 34.1 mmol) and propargyl alcohol (2.29 g, 40.9 mmol) were heated for 4 h at reflux in C6H6 (120 cm3) in the presence of a catalytic amount of p-TsOH (0.32 g, 1.7 mmol) using a Dean-Stark apparatus. The reaction was allowed to cool to room temperature and was washed with saturated NaHCO3 (2×100 mL) and then with water (2×100 mL). The organic layer was collected and dried (with anhydrous Na2SO4), filtered and concentrated to yield the crude product as an odorless mobile yellow oil (1.67 g, 50%) which was used without further purification.
Preparation of the [1-13C]-Hyperpolarized Acetate
The parahydrogenation reaction of the obtained propargyl ester has been carried out in an hydroalcoholic medium as formerly described in Example 1 for the acetate molecule and as schematized in scheme b) of
Synthesis of Propargyl Pyruvate
Pyruvic acid (3.00 g, 34.1 mmol) and propargyl alcohol (2.29 g, 40.9 mmol) were heated for 4 h at reflux in C6H6 (120 cm3) in the presence of a catalytic amount of p-TsOH (0.32 g, 1.7 mmol) using a Dean-Stark apparatus. The reaction was allowed to cool to room temperature and was washed with sat. NaHCO3 (2×100 mL) and then with water (2×100 mL). The organic layer was collected and dried (anh. Na2SO4), filtered and concentrated to yield the crude product as an odorless mobile yellow oil (2.08 g, 49%) which was not purified further.
Preparation of the [1-13C]-Hyperpolarized Pyruvate
Preparation of the [1-13C]-Hyperpolarized Allyl-Ester in Aqueous Medium
The parahydrogenation of the propargyl-pyruvate has been carried out in an hydro-alcoholic hydrogenation medium (15% methanol in water) using the water soluble catalyst [Rh(NBD)phos][BF4] (2.5 mM deuterated water solution).
The parandyrogenation reaction has been carried out into a NMR tube equipped with Young valve charged with 0.3 ml of catalyst, 0.03 mmol of substrate (3 μl, concentration about 70 mM), 0.05 ml of methanol and 8 bar of parahydrogen (enriched at 77K, 50% enrichment).
After warming the solution at 90° C., the NMR tube was vigorously shaken for 10″, then was dropped into a μ-metal magnetic field shield and successively slowly extracted from the shield in about 5″. The 13C-NMR spectrum has then been immediately acquired into a 600 MHz Bruker spectrometer. [1-13C]-hyperpolarized allyl pyruvate is obtained as schematized in
In the 13C spectrum acquired immediately after application of field cycling (shown in
Preparation of the [1-13C]-Hyperpolarized Allyl-Ester in Methanol
The parandyrogenation reaction has then been carried out in methanol, as reaction solvent, using the commercial catalyst [Rh(COD)dppb][BF4] (COD=cyclo-1,5-octadiene, dppb=1,4-bis(diphenylphosphino)butane). The parahydrogenation reaction has been carried out into a NMR tube equipped with Young valve charged with 2 mg of catalyst and 0.4 ml of methanol-d3. The catalyst was activated by hydrogenation of the coordinated COD, then 3 μl of substrate were added and the NMR tube was pressurized with 8 bar of parahydrogen (enriched at 77K, 50% enrichment).
After warming the solution at RT, the NMR tube was vigorously shaken for 10″, magnetic field cycling was applied as described in example 1 and then the 13C spectrum has been acquired. As can be observed from recorded spectrum, shown as
Preparation of the [1-13C]-Hyperpolarized Allyl-Ester in Methanol/CDCl3
The parahydrogenation reaction of the propargyl-pyruvate was then carried out in an organic medium consenting to benefit from a phase transfer-extraction of the [1-13C]-hyperpolarized pyruvate, allowing to remove the hydrogenation catalyst and to reduce to substantially non-detectable values the amounts of optional alcoholic (or water miscible) co-solvents in the final aqueous mixture, thereby leading to a substantially impurity-free aqueous solution of the desired [1-13C]-hyperpolarized pyruvate.
More particularly, the parahydrogenation reaction of the propargyl-pyruvate was carried out in a methanol/CDCl3 mixture (75 μl methanol and 500 μl CDCl3) with the commercial [Bis(diphenylphosphinobutane)(1,5-cyclooctadiene)]Rh(I) as hydrogenation catalyst. The parahydrogenation reaction has been carried out into a NMR tube equipped with Young valve charged with 2 mg of catalyst and 0.05 ml of methanol-d3. The catalyst was activated by hydrogenation of the coordinated diene, then CDCl3 and the substrate (3 μl of the propargyl ester in 25 μl of methanol) were added, the tube was pressurized with 8 bar of para-H2. After warming the solution at 90°, the NMR tube was vigorously shaken for 10″ to give the parahydrogenated intermediate of
The obtained basic solution was then brought to acidic pH (pH 4) by addition of 0.5 ml DCl. The aqueous phase was then collected and a 13C spectrum is immediately recorded shoving the 13C polarized signal of the pyruvate (
In order to may use the collected aqueous solution of hyperpolarized pyruvate in in vivo applications it is necessary that the aqueous solution is suitably buffered at a physiological pH (pH˜7). However, the basic solution of the hyperpolarized pyruvate (obtained after addition of NaOD (1M)) is, preferably first acidified to acidic pH, for instance by addition of DCl, as above said, consenting to restore the methyl group of the pyruvate molecule from α-deprotonated pyruvate and its corresponding hydrate form optionally formed at basic (pH>8) hydrolytic conditions. Then, the acidic solution is suitably buffered at pH˜7, for instance with phosphate buffer 0.1 M, thereby obtaining an impurity free, and physiologically acceptable solution of the hyperpolarized pyruvate ready for use in in vivo applications.
a) Synthesis of the Substrate Molecules
To a stirred suspension of 2 g (25.5 mmol) of glycine in 6 mL of a 30% w/w solution of sodium methoxide in methanol, 10 mL (51.10 mmol) of ethyl trifluoroacetate were slowly added at 0° C. Then temperature was slowly increased to room temperature and the solution was stirred for 4 hours. Then methanol was evaporated and the residue was partitioned between aqueous HCl 1M and diethyl ether. The organic phase was extracted with diethyl ether (2×100 mL) and the combined organic layers were washed with brine (3×100 mL), dried over Na2SO4 and the solvent was evaporated under reduced pressure. A white solid was obtained (3.61 g, 79%) and it was used with no further purification.
NMR characterization: 1H NMR (acetone-d6 400 MHz) d 8.90 (1H, bs, NH), 4.06 (2H, d, J=5.86 Hz, CH2)
13C NMR (acetone-d6, 100 MHz) d 169.7 [C, COOH], 158.0 [C, q, 2JC—F=36.8 Hz, COCF3], 117.0 [C, q, JC—F=285.3 Hz, COCF3] 41.4 [CH2].
19F NMR (acetone-d6 376 MHz) d −76.44
To a stirred suspension of the intermediate compound obtained from step i) (1.30 g, 7.60 mmol) in 15 mL of vinyl acetate, Pd(OAc)2 (0.02 g, 0.08 mmol) and KOH (0.04 g, 0.76 mmol) were added under argon atmosphere. The solution was stirred overnight at room temperature. The mixture was then quenched with water and diethyl ether was added. The aqueous phase was extracted with diethyl ether (2×50 mL), washed with brine (3×50 mL), dried over Na2SO4, filtered and the solvent was evaporate. The crude reaction was then purified on silica gel using diethyl ether/petroleum ether (40/60) as eluent to afford the desired product as a pale yellow solid (0.37 g, 25%).
NMR characterization:
1H-NMR (acetone-d6 400 MHz) d 9.00 (1H, s, NH), 7.26 (1H, dd Jcis=6.16 Hz, Jtrans=13.76 Hz CH), 4.93 (1H, Jtrans=13.76 Hz, CH2), 4.68 (1H, d, Jcis=6.16 Hz, CH2), 4.25 (2H, d, J=4.25, CH2)
13C-NMR (acetone-d6, 100 MHz) d 166.6 [C, COOVinyl], 158.3 [C, 2JC—F=36.7 Hz, COCF3], 141.8 [CH], 116.9 [C, JC—F=285.2 Hz, COCF3], 99.0 [CHCH2], 41.5 [NHCH2]
19F NMR (acetone-d6 376 MHz EK 492) d −76.5
b) Parahydrogenation of the Trifluoro-Acetyl Glycine Vinyl-Ester in Acetone and Methanol.
The hydrogenation reaction of the trifluoro-acetyl vinyl-glycine 1 obtained as above described was carried out either in acetone or in methanol according to the following reaction scheme
by using, with both solvents, the commercial hydrogenation catalyst [Rh(COD)dppb] and by following, in both cases, the procedure already used for preparation of the [1-13C]-hyperpolarized allyl-ester in methanol, as described in Example 3. Following to the application of field cycle, a 13C polarized carbonyl signal (167 ppm) was observed in both solvents, as observed, for instance, in the 13C-NMR spectrum of
Under argon atmosphere, 0.2 mL (3.51 mmol) of propargyl alcohol and 0.02 g (0.18 mmol) of DMAP ((Dimethylamino)pyridine) were added to a stirrer solution of compound 1 in dry dichloromethane. Temperature was decreased at 0° C. and 2.63 g (2.63 mmol) of DCC (Dicyclohexylcarbodiimide) were added. The temperature was slowly increased until room temperature and the solution as stirred for 3 hours. The white formed precipitate was filtered off and dichloromethane was evaporated under reduced pressure. The crude was purified on silica gel using diethyl ether/petroleum ether (50:50) as eluent affording 0.25 g (68%) of the desire product as a white solid.
NMR characterization:
1H-NMR (acetone-d6 400 MHz, EK 473) d 8.89 (1H, bs, NH), 4.79 (2H, d, J4=2.36 Hz, COOCH2), 4.17 (2H, d, J=5.88, NHCH2), 3.05 (1H, t, J4=2.36 Hz, CH)
13C-NMR (acetone-d6, 100 MHz, EK 473) d 168.4 [C, COO-propargyl], 158.3 [C, 2JC—F=36.7 Hz, COCF3], 116.9 [C, q, JC—F=285.2 Hz, COCF3], 78.2 [C, CCH], 76.8 [CH, CCH], 53.3 [CH2, COOCH2], 41.5 [CH2, NHCH2]
19F NMR (acetone-d6 376 MHz EK 492) d −76.4
The parandyrogenation of the obtained TFA-Gly-OPropargyl 1 has been carried out using the commercial catalyst [Rh(COD)dppb] in a Methanol/CDCl3 mixture (200μ/500 μl) using the same procedure described in Example 3 for the preparation of the [1-13C]-hyperpolarized allyl-ester in methanol/CDCl3, with 200 μl of methanol-d3 instead of 75 μl. Following to the application of field cycle, the hydrolysis of the [1-13C]-hyperpolarized TFA-Gly-OAllyl ester has been performed by the addition of 1 ml NaOD 2M consenting to remove both the hydrogenated ester moiety (namely allyl-alcohol) and the protecting group TFA (trifluoroacetic acid). A 13C-NMR spectrum of the directly collected aqueous phase was then recorded, shown in
To a stirring suspension of 2-Acetoxy-propionic acid (3.0 g, 23 mmol) in 66 mL of vinyl acetate, Pd(OAc)2 (0.076 g, 0.34 mmol) and KOH (0.200 g, 3.5 mmol) were added under nitrogen atmosphere. The solution was stirred for 48 hours at room temperature. The mixture was then quenched with water, the catalyst was filtered and diethyl ether was added. The aqueous phase was extracted with diethyl ether (3×40 mL), washed with brine (3×50 mL), dried over Na2SO4, filtered and the solvent was evaporate. The crude was then purified on silica gel using diethyl ether/petroleum ether (1/4) as eluent to afford the desired product as a pale yellow oil (0.579 g, 16%).
1H-NMR (CDCl3 400 MHz) 7.2 (1H, dd, CH2), 5.1 (1H, q, CH) 4.95 (1H, d CH), 4.65 (1H, d CH2), 2.12 (3H, s, CH3), 1.5 (3H, d, CH3).
13C-NMR (CDCl3, 100 MHz) d 171 [C, COOVinyl], 168[C, COOCH3], 141 [CH], 100 [CHCH2], 67 [CH], 20 [CH3], 17 [CH3]
The parahydrogenation of 2-Acetoxy-propionic acid vinyl ester (1) has been carried out in water with the water soluble catalyst [Rh(NBD)phos][BF4] according to the same procedure reported in Example 1 for the preparation of the [1-13C]-hyperpolarized ethyl ester of acetate, using 0.05 ml of acetone instead of methanol.
Following to the application of magnetic field cycling, a 13C polarized carbonyl signal (173 ppm) of 2-Acetoxy-propionic acid ethyl ester (3) was observed (
To get the corresponding [1-13C]-hyperpolarized lactate, the para-hydrogenation reaction is repeated as above described with a second amount of vinyl ester (1) and the obtained [1-13C]-hyperpolarized ester is hydrolysed by addition of 0.05 ml of NaOD 12M immediately after field cycling procedure. The obtained solution is then acidified by the addition of 0.05 ml of DCl 12M and a 13C-NMR spectrum is immediately acquired.
| Number | Date | Country | Kind |
|---|---|---|---|
| 13190409.6 | Oct 2013 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2014/072983 | 10/27/2014 | WO | 00 |
