ITMI20081765A1 - PROCESS FOR THE PREPARATION OF HYPERPOLARIZED MOLECULES. - Google Patents

Description

DESCRIPTION

The present invention relates to the technical field of diagnostic visualization by magnetic resonance, also known as â € œMagnetic Resonance Imagingâ € or also MRI. More particularly, the present invention relates to a process for the preparation of hyperpolarized molecules by catalytic parahydrogenation, and their subsequent isolation by transfer in aqueous phase.

This makes it possible to advantageously obtain an aqueous solution of the hyperpolarized molecule to be used in the above diagnostic techniques, since said molecule is purified from the catalyst, the organic solvent and any unreacted starting compound, in a single step.

State of the art

Magnetic Resonance Imaging (MRI) is known to be a powerful tool for medical and biological investigations, both in vitro and in vivo. However, the main disadvantage of this technique is due to the low sensitivity of the NMR spectroscopy on which MRI is based. In fact, the intensity of the NMR signals depends on the population difference between the nuclear spin states, in turn a function of the temperature and of the applied magnetic field, according to the well-known Boltzmann equation (Î "Î = yhBo / (2nkT) ), at thermal equilibrium, said population difference is in the order of magnitude equal to 10 '<5>, that is to say very small.

The use of hyperpolarized molecules in magnetic resonance imaging (MRI) has recently been proposed as a possible solution to this problem and, in recent years, several methods of hyper polarization have been developed.

The most direct method is the so-called â € œbrute forceâ € method which essentially consists in keeping the molecule of interest inside a high magnetic field (up to 20 T) and at a very low temperature (close to 0 K) to a certain period of time. This method is generally applicable but requires the application of an appropriate â € œrelaxation switchâ € that allows to quickly promote the nuclear transitions necessary to create polarization, and that can be removed or in any case â € œdeactivatedâ € immediately after the process. . At present, a suitable â € œrelaxation switchâ € is not yet available and, consequently, this approach is not yet usable.

A second method is represented by the so-called â € optical pumping / spin exchangeâ €, which can be applied to noble gases such as <129> Xe and T <3> He. In this case, a beam of polarized laser light is sent onto a gaseous mixture consisting of the selected gas and vapors of an alkaline metal. In this way it is possible to hyperpolarize Xe and He with high degrees of polarization, and the polarization thus obtained can then be maintained for a long time thanks to the long relaxation times of these nuclei.

In this regard, MRI studies of the airways conducted with the use of hyperpolarized gases thus obtained are known.

However, as previously indicated, this technique is not of general applicability as it is limited to the polarization of noble gases.

The population difference between the nuclear spin levels can also be increased by exploiting the â € œOverhauserâ € effect (or Nuclear Dynamic Polarization - DNP) between the nucleus of interest and the unpaired electrons of paramagnetic species in contact.

This technique is used to hyperpolarize molecules of biological interest such as, for example, urea, pyruvate etc., currently used for metabolic imaging studies by MRI.

Although this technique can be applied to any type of molecule, in principle, the need for a powerful cryostat and suitable â € œ hardwareâ € for the irradiation of electrons at low temperatures can limit its use.

Furthermore, a procedure is required that allows the rapid dissolution of the substrate after hyperpolarization, and the separation of the paramagnetic radical before injection.

More recently, in the diagnostic visualization by MRI, the use of hyperpolarized molecules by means of parahydrogenation of unsaturated substrates in the presence of para-hydrogen has been proposed (US 6,574,495). This procedure is also known as PHIP (Para Hydrogen Induced Polarization), or Polarization Induced by Para-Hydrogen. In this regard, the PHIP method allows to obtain deeply altered populations of nuclear spin levels compared to those determined by Boltzmann thermodynamics. The hyperpolarized molecules are obtained by catalytic hydrogenation of an unsaturated substrate using hydrogen enriched in the spin para isomer. The hydrogen molecule exists, in fact, in the two isomeric forms called ortho and para respectively: while the ortho isomer, symmetrical with respect to the exchange of the two protons, is triply degenerate, the para isomer, antisymmetric with respect to the exchange , Is in a singlet state. The ortho isomer has spin 1 and is NMR active while the para isomer, having spin zero, is silent NMR.

The equilibrium mixture (normal-hydrogen) contains 75% of ortho isomer and 25% of para isomer but, being the para thermodynamically more stable state and following the relatively high rotational temperature of the hydrogen molecule, it is possible to enrich the mixture in the para form, keeping it at a low temperature.

For example, if at 77 K the equilibrium mixture is made up of 52% para isomer and 48% of ortho isomer, at 20 K it is made up of 99.8% para isomer.

Conversion between the two isomers is prevented for reasons of symmetry but can be promoted by the presence of a suitable catalyst, such as activated carbon or iron oxide.

The enrichment in the para isomer obtained at low temperature can be kept at room temperature as long as the catalyst that promoted the conversion, and any other paramagnetic impurity, are totally removed. This procedure therefore allows to obtain hydrogen mixtures in non-equilibrium conditions, at room temperature, and the mixture thus enriched is called para-hydrogen.

Although NMR is silent, when para-hydrogen is added to an unsaturated molecule its symmetry can be broken with the formation of an AX-type spin system, generating the possibility of observing hyperpolarization.

The populations of the spin levels of the parahydrogenated products obtained differ from the equilibrium, thus making it possible, theoretically, an increase in sensitivity by a factor equal to IO <5> (â € œSensitive enhancement utilizing para-hydrogenâ €, C.R. Bower, Encyclopedia of NMR, Vol. 9, 2002, pp. 750-770).

However, for the purposes of in vivo diagnostic visualization using magnetic resonance, heteronuclear polarization is more useful as the proton signals of a parahydrogenated contrast agent would overlap with the <J> H signals of tissue water while, on the contrary, the endogenous signal for the heteronucleus is practically 0 so the absence of background noise allows to obtain images with an extremely favorable signal-to-noise ratio, where the contrast is given by the difference in signal intensity between regions in which is present the molecule comprising hyperpolarized Teteronucleus and areas in which it is absent.

The attention was therefore focused on heteronuclei such as <13> C and <I5> N. An advantage in the use of these species, in addition to the already mentioned absence of background noise, is associated with their large range of Chemical shift, which offers the possibility to visualize different molecules in the same image.

Using the PHIP hyperpolarization method it is possible to obtain hyper polarized <13> C and <15> N molecules. In order to obtain a hyperpolarized <13> C molecule that can be advantageously used in vivo in diagnostic imaging by MRI, however, the following requirements must be met:

1) The substrate must be easily hydrogenable,

2) The substrate must contain a nucleus <13> C within a distance of three bonds from the proton added with the para-H2 molecule,

3) The molecular weight of the hyperpolarized molecule must be low (<500 Da), to limit the relaxation processes,

4) The hydrogenated product must naturally be physiologically well tolerable and soluble in water,

5) The catalyst used for the para-hydrogenation must promote the transfer of both protons, from a molecule of B.2 to the same substrate molecule, so that the spin correlation can be maintained. Consequently, homogeneous catalysts such as, for example, the organometallic complexes of Rh and Ir must be used. However, as these are highly toxic species, these catalysts must be completely removed from the reaction mixture prior to in vivo administration.

6) For in vivo application an aqueous solution of the para-hydrogenated product must be available. This therefore requires that the hydrogenation reaction is carried out in water or, alternatively, that the organic solvent used for the hydrogenation is then totally removed,

7) In order to be used for the acquisition of an MRI image, the spin order of the para-hydrogen must be transformed into net magnetization <13> C. It has been shown that this can be achieved by applying a suitable field cycle to the para-hydrogenated product, or by using a suitable pulse sequence.

Similar considerations apply to the preparation of hyperpolarized molecules containing other nuclei that can be hyperpolarized with this procedure.

Therefore a problem connected to the in vivo use of hyperpolarized compounds is linked to the need for them to be available in aqueous solution while, generally, the hydrogenation reaction with para-hydrogen of a suitable unsaturated substrate is carried out in the presence of a solvent organic in which both the hydrogenation catalyst and the hydrogen itself are more soluble.

It is known that the catalysts which best favor the transfer of the polarization to <13> C, after the addition of a molecule of para-hydrogen to unsaturated substrates, are those constituted by the cationic Rh (I) complexes. Preferred are catalysts which contain a chelating phosphine, for example dppb (diphenylphosphinobutane) or dppe (diphenylphosphinoethane), and a diene molecule such as, for example, cyclooctadiene or norbornadiene.

These catalysts have the best efficiency in organic solvent, where acetone is preferred.

Attempts have been made to render these catalysts more soluble in aqueous solvent, for example by introducing ionic / polar groups on the phosphmic ligands of the chelant itself. On the other hand, an important drawback that must be faced when carrying out the hydrogenation reaction in water is linked to the low solubility of hydrogen in water, which therefore makes it necessary to operate at high pressures (50-100 bar).

Furthermore, since these are highly toxic compounds, these catalysts must be completely removed from the reaction mixture at the end of the hydrogenation. One method for removing the cationic Rh complex from the reaction mixture consists in passing it over a suitable cation exchange resin, although this procedure involves a marked loss of polarization.

Alternatively, the use of Rh (I) based catalysts supported on a solid surface based on silica or polymers has been proposed. However, the polarization obtainable with this type of supported catalysts is lower than that observed with homogeneous catalysts, probably due to the lower mobility of the substrate / catalyst adduct which generates an increase in the relaxation rate.

Of course, the organic solvent must also be removed from the hyperpolarized solution prior to its formulation in aqueous solvent intended for administration. This objective can be achieved, for example, through the use of a â € œ spray-drierâ € placed immediately downstream of the reactor: the process is similar to the â € œspraydryingâ € commonly used to transform a solution into solid particles, for pharmaceutical compositions. The fluid material is thus introduced into the â € œdrierâ €, where it is nebulized and dispersed by a carrier gas: the more volatile solvent is then distilled by applying a vacuum while the water, previously added to the mixture, remains in the â € œdrierâ €. A drawback associated with this procedure derives from the fact that a large part of the parahydrogenated product (low molecular weight molecule) can be lost together with the organic solvent.

It therefore remains of particular importance the possibility of finding a procedure for the preparation of hyperpolarized molecules, in aqueous solution ready for use, in the diagnostic visualization of organs and tissues of the human body using MRI techniques and which allows, at the same time, to overcome the drawbacks and purification problems described above.

Object of the invention

The present invention refers to a process for the preparation of hyperpolarized molecules ready for use which, advantageously, does not require the filtration of the catalyst, the purification of the product and the subsequent formulation of the same in aqueous solution.

In particular, the present invention relates to a process for the preparation, in a single step, of hyperpolarized molecules in aqueous solution ready for use for the diagnostic visualization of organs or tissues of the human or animal body, by means of Nuclear Magnetic Resonance ( MRI).

Even more particularly, the present invention relates to a process for the preparation of hyper polarized molecules which allows to isolate in a single step, by means of a phase transfer process, the hyperpolarized molecule and to obtain it directly in aqueous solution, free of impurities and ready for use, for in vivo diagnostic visualization by magnetic resonance imaging.

Preferably, according to the process object of the present invention, the hyperpolarized molecules are obtained by hydrogenation with para-hydrogen of a suitable unsaturated substrate which is soluble in an organic solvent.

Organic solvents suitable for the purpose include organo-chlorinated solvents such as, for example, chloroform, dichloromethane, carbon tetrachloride, etc., aromatic solvents such as, for example, benzene and toluene, ethers such as, for example, diethyl ether, diisopropyl ether, butyl ether etc., aliphatic hydrocarbons such as pentane, hexane, heptane, octane, cyclohexane, etc.

Among these, the chlorinated solvents and hydrocarbons listed above are preferred.

Even more preferred are chloroform and dichloromentane. Said hydrogenation reaction is preferably carried out in the presence of a suitable catalyst, which must be soluble in organic solvent but insoluble in water.

Examples of suitable hydrogenation catalysts include [Rh (diphosphma) {diene)] <+> [anion] -, where diphosphine can be DPPB (1,4-diphenylphosphinobutane), DPPE (1,2-diphenylphosphioethane) and their derivatives , including for example chiral phosphines such as BINAP {2,2â € ™ -Bis {diphenylphosphine) -1,1-binaphthyl), CHIRAPHOS (2,3-diphenylphosphinobutane), DIOP {1, 4-Bis (diphenylphosphine) - 1,4-dideoxy-2,3-0-isopropylidene-L reitol), DIPAMP (1,2-Bis [(2-methoxyphenyl) (phenylphosphino)] ethane); the diene can be 1,5-cyclooctadiene or norbornadiene, and the anion can be any anion even if the tetrafluoborate or trifluoromethyl sulfonate are to be considered preferred.

Among these, the catalysts in which the phosphine is diphenylphosphinobutane and particularly preferred is [Bis (diphenylphosphinobutane) (1,5-cyclooctadine)] Rh (I) are to be considered preferred.

According to a particularly preferred aspect, the present invention relates to a process in which:

a) a suitable unsaturated substrate is solubilized in an organic solvent immiscible with water and hydrogenated, with parahydrogen, in the presence of a catalyst soluble in the organic solvent but insoluble in water,

b) the hyperpolarized molecule thus obtained is isolated from the (crude) reaction system, by simple dilution of the same with water or with a suitable aqueous solution, and by subsequent separation of the aqueous phase containing the hyperpolarized product.

By aqueous solution we mean a suitable saline solution, possibly suitably buffered, however physiologically acceptable and usable in vivo in diagnostic imaging without further purification.

Particularly preferred, for the purpose of the present invention, are to be considered unsaturated substrates which are well soluble in an organic solvent and less soluble in water, and whose corresponding parahydrogenated molecules present, on the other hand, an increased solubility in water. In this way the hydrogenation reaction is carried out in an organic solvent that is not miscible with water, and the hyperpolarized molecule is transferred to the aqueous phase following the addition of water, essentially as shown in Figure 1.

It is also understood that the preferred unsaturated substrates according to the invention contain a double or triple C-C bond, which is reduced during the addition of para-hydrogen.

According to a particularly preferred aspect, the present invention relates to a process for the preparation of heteronuclear hyperpolarized molecules where <13> C and <15> N are particularly preferred. In this case, it is advisable to favor the transfer of the polarization from the protons of the para-hydrogen to the heteroatom in question.

For the use of hyperpolarized derivatives by means of parahydrogenation as contrast agents for <13> C-MRI, in fact, it is necessary to convert the signal into an anti-phase of the hyperpolarized carbon, which is obtained following the transfer of polarization from the protons of the parahydrogen to the carbon in question, in a phase signal, useful for image acquisition. This can be achieved: 1) by recording an MRI image using an appropriate sequence of pulses (see Goldman M., Johannesson H., C.R.Phisique 2005, 6, 575), or 2) by applying a field cycle to the hydrogenated product. The field cycle consists quickly (non-adiabatically) the hydrogenated sample inside a magnetic field screen (field strength = 0.1 Î1⁄4Τ), and then slowly (adiabatically) removing the screen to bring the sample back on a terrestrial field (50 Î1⁄4Τ).

Therefore, in a particularly preferred aspect the present invention refers to a process in which:

a) a suitable unsaturated substrate is solubilized in an organic solvent immiscible with water and hydrogenated, with parahydrogen, in the presence of a catalyst soluble in the organic solvent but insoluble in water,

b) a suitable field cycle is applied to the parahydrogenated product,

c) the hyperpolarized molecule thus obtained is isolated from the (crude) reaction system, by simple dilution of the same with water or with a suitable aqueous solution, and by subsequent separation of the aqueous phase containing the hyperpolarized product.

In the event that the process of the invention is used for the preparation of hyperpolarized molecules with heteronuclei, in particular at <13> C, in the substrate molecule there must also be a carbon atom within two or three bonds from the protons derived from the parahydrogen which, preferably, is characterized by a long relaxation time.

Particularly preferred among the latter are, for example, the carbon atoms belonging to carbonyl groups or the quaternary carbon atoms.

Advantageously, operating according to the process object of the present invention, the scalar coupling between the added protons and <13> C allows the transfer of polarization to the heteronucleus, while the long relaxation time allows to maintain the polarization for a few tens seconds, and even more preferably for more than 60 seconds, in physiological conditions.

Finally, the group containing carbon atom to which to transfer the polarization should be enriched in <13> C. For in vivo imaging applications it is preferable that the degree of enrichment is 99% or greater.

Among the possible substrates that can be used according to the process object of the present invention, labeled in <13> C, <15> N or other heteronucleus having a nuclear spin 1⁄2, the substituted alkynes are preferable which, by partial saturation, generate the corresponding substituted alkenes ; preferably, the difference in solubility in water between the starting alkene and the alkene thus produced is at least equal to 60%, naturally in favor of the alkene characterized by greater solubility.

Equally preferred are, in addition, the substituted alkenes which generate the corresponding substituted alkanes, with a difference in solubility in water preferably equal to 60% in favor of the reaction product (alkane) therefore characterized by a higher solubility.

The starting products according to the process object of the present invention, that is the unsaturated substrates susceptible to parahydrogenation, preferably marked in <13> C, <15> N or other heteronucleus having nuclear spin 1⁄2, are known compounds or easily prepared according to methods known.

Similarly, the catalysts used in the process in question are known or, if not commercially available as such, can be prepared with known methods.

In the same way, the suitable organic solvent immiscible with water can be chosen among the numerous examples of solvents known to the skilled in the art and usable in the hydrogenation reactions.

Eventually, the solvent system can also consist of a suitable mixture of solvents.

A further aspect of the present invention also contemplates the possibility of suitably modifying the solubility of the substrate, for example by reacting it with a suitable functional group capable of suitably increasing or decreasing its solubility in aqueous solvent.

Examples of functional groups that can be used for this purpose include: alcoholic, ethoxyl and polyethoxyl groups with different chain length, and benzoxyl, aminic, carboxylic groups. Among these, the preferred are the alcoholic, ethoxy and polyethoxyl groups which make the substrate more soluble in aqueous solution.

For example, it is possible to use advantageously groups such as the benzyloxy group, which on the one hand increase the solubility of the substrate to be hydrogenated in the organic reaction solvent and which, during the hydrogenation reaction can then be removed, for example by hydrogenolysis.

Description of the Figures

Figure 1: isolation scheme of the parahydrogenated product by phase transfer;

Figure 2: <13> C-NMR spectra (14 T, 298 K, acetone-d <6>), of B (enriched in <13> C) obtained from the para-hydrogenation of A. a) spectrum recorded immediately after para-hydrogenation and the field cycle; b) spectrum recorded after relaxation (5 minutes). S indicates the solvent, i an impurity.

Figure 3: <13> C-NMR spectra (14 T, 298 K, D2O), of B (enriched in <13> C) obtained from the para-hydrogenation of A. a) spectrum recorded immediately after para-hydrogenation, the field cycle and D2O extraction; b) spectrum recorded after relaxation (5 minutes).

In order to exemplify the process object of the present invention and to provide possible indications regarding the operative conditions that can be adopted, without these being intended as limitations to the process, the following examples are now provided.

Example 1

Alkyne A referred to in the formula below has been synthesized by transesterification of the corresponding dimethylacetylenedicarboxylate with diethylene glycol monomethyl ether, in the presence of H2SO4 as catalyst.

The oilyoxyethylene chains increase its solubility in water: it has therefore been verified that the solubility in water is greater for the hydrogenated derivative B (alkene) than for the starting substrate A (alkyne).

A P-H2

0 Q

H * <'>

B.

The unsaturated substrate A (0.02 mmol) was para- hydrogenated in a 5 mm diameter NMR tube equipped with a Young valve, in acetone-d6 (0.4 ml), in the presence of 5 mg of [Bis (diphenylphosphinobutane) ( 1,5-cyclooctadine)] Rh (I) tetrafluoborate (previously activated by reaction with 3⁄4) and 4 atm of para-H2 (52%).

The reaction was carried out by shaking the tube for 10 seconds (yield 85%), and the increase in the signal of the carboxylic group of the alkene obtained in the <13> C spectrum recorded immediately after was equal to 1500 times .

To obtain a <13> C in phase signal, useful for recording MR images, a field cycle (as reported for example in US patent 6,574,495, 2003, Golman et al.) Was applied to the sample. The field cycle was performed by quickly inserting the tube into a Î1⁄4-metal screen (magnetic field of the screen: 0.1 Î1⁄4Τ), and slowly removing the screen in order to bring the sample back to the Earth's magnetic field. The whole procedure takes 3-5 seconds. The tube was then inserted into the spectrometer (14 T) for the acquisition of the spectrum <13> C, which is shown in Figure 2 (increase in signal intensity equal to 250).

Example 2

The samples for the separation of the catalyst and the organic solvent by phase transfer were prepared by parahydrogenating compound A in a mixture of CDCI3 and acetone-d6 6: 1 (under the same conditions reported above).

The chloroform is immiscible with water, and a small quantity of acetone is however necessary to maintain a good efficiency of the catalyst. After carrying out the field cycle, 0.4 ml of previously degassed D2O were introduced into the NMR tube. The tube was shaken vigorously for 3 seconds, and then it was left stationary for 5 seconds, in order to allow the organic and aqueous phase to separate. The aqueous phase was then withdrawn with a syringe and transferred to another tube for observation. The resulting spectrum is shown in Figure 3.

The increase in intensity (100) of the <13> C signal is lower than that observed in pure acetone due to a partial loss of polarization that takes place during phase separation. However, the residual polarization is sufficient for the observation of an intensified signal corresponding to B dissolved in D2O (165.99 ppm). In the aqueous solution there is a trace of residual CDCI3 (7%), which gives rise to the signal at 164.82 ppm, relating to B dissolved in chloroform. The quantity of B transferred in the aqueous phase is estimated to be about 10% of the total.

Claims (10)

CLAIMS 1) A process for the preparation, in a single step, of hyperpolarized molecules in aqueous solution, ready to use for the diagnostic visualization of organs or tissues of the human or animal body including the isolation of the hyperpolarized molecule by phase transfer. 2) A process for the preparation according to claim 1 comprising the par-hydrogenation of a suitable unsaturated substrate, in the presence of a suitable catalyst and organic solvent immiscible in water and, by addition of water, the isolation of the corresponding para- hydrogenated by separation of the aqueous phase. 3) A process according to claim 1 where the unsaturated substrate is insoluble or scarcely soluble in water and the corresponding para-hydrogenated substrate is soluble in water. 4) A process according to claim 3 where the unsaturated substrate is constituted by a suitable alkenic or alkenic group optionally substituted and the para-hydrogenated substrate is constituted by the corresponding alkenic or alkanic derivative, respectively. 5) A process according to one of claims 1 to 4 where the unsaturated substrate is suitably marked in <13> C, <15> N or other heteronucleus having a nuclear spin 1⁄2. 6) A process according to one of the preceding claims where the catalyst is [Bis {diphenylphosphinobutane) (1,5-cyclooctadine)] Rh (I). 7) A process according to one of the preceding claims where the water-immiscible organic solvent system comprises an organochlorine solvent, an aromatic, ethereal solvent, or an aliphatic hydrocarbon. 8) A process according to one of the preceding claims in which: a) a suitable unsaturated substrate is solubilized in an organic solvent immiscible with water and hydrogenated with parahydrogen, in the presence of a catalyst soluble in the organic solvent but insoluble in water, b) the parahydrogenated molecule thus obtained is isolated from the raw reaction product by diluting it with water or an aqueous solvent, by separation of the aqueous phase containing the hyperpolarized product. 9) A process according to one of the preceding claims where the hyperpolarized molecule is physiologically tolerable. 10) A process according to claim 9 for the preparation, in a single step, of hyperpolarized molecules in aqueous solution ready to use for the diagnostic visualization of organs or tissues of the human or animal body by means of Nuclear Magnetic Resonance.