As the phosphonate groups of the pendant arms were found to be responsible for the pH responsive nature of Gd1
] it was important to maintain this structural feature when the complex was modified to facilitate attachment to the dendrimer. Accordingly, the complex was modified by incorporating a functionalized benzyl group onto the macro-cyclic backbone of the complex, leaving the four phosphonate groups intact. The functionalized complex Gd2
was prepared following the same synthetic procedure used for the preparation of Gd1
] simply substituting (S
-nitrobenzyl) cyclen for cyclen ().
Synthesis of a functionalised pH responsive contrast agent. i) P(OEt)3/Δ ii) H2NNH2/EtOH; iii) BrCH2COBr/K2CO3/CH2Cl2; iv) (S)-2-(p-nitrobenzyl) cyclen/K2CO3/MeCN/60°C; v) 30% HBr in AcOH.
was prepared by the Michaelis–Arbuzov reaction of triethylphosphite with bromomethyl phthalimide. The key intermediate 4
was then obtained by removal of the phthalimide protecting group by an Ing–Manske[24
] reaction with 1.2 equivalents of hydrazine in ethanol. Normally this type of deprotection reaction proceeds in good yields;[24
] however, in our case the yields were moderate at best.[12
] Furthermore, the presence of significant quantities of a reaction by-product meant that column chromatography was necessary to purify amine 4
, an oil at room temperature. Amines can often be purified by an acid/base extraction but 4
shows good water solubility, even at high pH, and this precluded purification by extraction. Low yielding reactions that require complicated purification procedures are unsuitable for the scale-up necessary for the production of large quantities of an MRI contrast agent. We therefore undertook an investigation into the reasons for the low reaction yield of this step.
H NMR analysis of the crude reaction mixture indicated that the primary contaminant of the product 4
was a single reaction by-product. This compound was isolated by column chromatography and characterized. Rather surprisingly, this by-product was identified as compound 5
, the product of a reaction between the starting material 3
and the intended reaction product 4
(). A significant effort has been applied to understanding the mechanism and intermediates of the Ing–Manske reaction,[25
] however, this particular reaction pathway is rarely included in these discussions.[29
] The quantities of 5
produced suggest that this side reaction may, on occasion, be more important than generally described. The mechanism of the Ing–Manske reaction is quite complex[26
] but it is initiated by nucleophilic attack of hydrazine at one of the carbonyls of the phthalimide. The effectiveness of an amine at removing phthalimide protecting groups has been shown to relate to the protonation constant of the attacking amine; the more basic the amine, the more effective the reaction.[27
] Thus, if the product amine is significantly more basic than hydrazine, it is likely to compete with hydrazine in attacking the phthalimide 3
to yield significant quantities of compound 5
The identity of compound 5
was established by NMR spectroscopy and mass spectrometry and, although attempts to remove compound 5
from the crude reaction mixture by crystallisation were unsuccessful, compound 5
readily crystallises once purified. X-ray quality crystals could be grown at room temperature from a solution of 5
in dichloromethane by addition of diethyl ether and hexanes. This allowed the structure of compound 5
to be confirmed by X-ray crystallography (). The production of compound 5
during the deprotection of the phthalimide 3
is doubly detrimental to the yield of 4
since two molecules of 4
are taken up into one molecule of 5
; this is in addition to the need for chromatography. Thus, eliminating compound 5
from the reaction would both improve yield and simplify purification since the other reaction by-product, the insoluble phthalhydrazide, can be removed by filtration. A number of improvements to the Ing–Manske reaction have been suggested, most notably raising the reaction pH through addition of sodium hydroxide.[26
] However, concerns over the lability of the phosphonate diester moiety towards base-catalysed hydrolysis precluded this as a solution to this problem. We investigated a number of the other Ing–Manske reaction conditions to assess whether the reaction conditions could be improved.
ORTEP rendering of the crystal structure of 5 showing 50% ellipsoids. Hydrogen atoms have been omitted for clarity.
Compounds 4 and 5 are easily distinguished by 1H NMR spectroscopy; a shift difference of almost 1 ppm is observed for the methylene resonance α to the phosphorous. This resonance appears as a doublet at 2.9 ppm (coupled to phosphorous) in 4 and as a doublet of doublets at 3.8 ppm (coupled to phosphorous and the amide proton) in 5. A series of experiments were performed in which various reaction conditions were altered and the ratio of the two reaction products in the crude reaction mixture determined by 1H NMR spectroscopy. Neither increasing the reaction temperature from room temperature to reflux nor increasing the reaction time from 6 h to 5 d was found to have a significant effect upon the distribution of reaction products. However, when the amount of hydrazine used in the reaction was increased the amount of 4 obtained from the reaction was greatly increased at the expense of 5. Increasing the amount of hydrazine from one to four equivalents greatly reduced the amount of 5 produced in the reaction. At six equivalents of hydrazine the yield of 5 was < 5%, and ten equivalents of hydrazine eliminated all traces of 5 from the reaction 1H NMR spectrum. This observation does not indicate that 5 is not produced during the reaction. Indeed, when an purified sample of 5 was treated with ten equivalents of hydrazine it was found to undergo a quantitative conversion to 4, as determined by 1H NMR (). Thus, 5 may still be produced in the reaction but, importantly, it is reactive under these conditions and does not persist as a reaction product.
Changing the conditions of the Ing–Manske reaction by increasing to ten the equivalents of hydrazine allowed purification to be simplified from column chromatography to filtration to remove the phthalhydrazide. It also improved the reaction yield to 97% and rendered this process suitable for scale-up. Condensation of the amine 4 with bromoacetyl bromide afforded the bromoacetamide 6 which was used to alkylate (S)-2-(p-nitrobenzyl) cyclen in acetonitrile with K2CO3 as base (). Subsequent deprotection of the phosphonate esters of 7 with HBr in AcOH afforded the ligand 2 a functionalized analogue of 1 that preserved the integrity of the four phosphonate groups.
It was later found that conjugation of the bifunctional ligand to the PAMAM dendrimer was more efficient if the protected ester ligand 7 was used instead of the free acid. Reduction of the nitro group with hydrogen and palladium catalyst afforded the corresponding amine 8 in 72% yield (). The amine was then converted to the isothiocyanate 9 by reaction with thiophosgene in a biphasic reaction at pH 2. The isothiocyanate group is ideal for conjugation with primary amines, such as those that decorate the surface of the PAMAM dendrimer, under mild conditions. The phosphonate ester was preferred for this conjugation reaction in order to minimize non-reactive salt formation between the amines of the dendrimer and phosphonic acids and repulsion between conjugated and incoming phosphonates. The ethylenediamine core G5-PAMAM dendrimer selected as the basis of our macromolecular construct has 128 primary amine groups on its surface. The dendrimer was reacted with 256 equivalents of 9 for 24 h at 40 °C followed by a further 256 equivalents for 48 h. The reaction pH was maintained at 9 throughout by addition of a 1m solution of NaOH. The reaction was analysed by HPLC using a Phenomenex BIOSEP SEC S-3000 size exclusion column (5–700 kD, PBS buffer, pH 7.4). The chelate–dendrimer conjugate was purified by repeated diafiltration using a Centricon C-30 membrane with a 30 kD cut-off (Millipore) until no low molecular weight materials could be detected by HPLC. HPLC analysis of the resulting dendrimer indicated that an average hydrodynamic volume equating to a molecular weight of about 140 kD was achieved. This corresponds to an average of 75% coverage or 96 ligands per dendrimer. A ligand/dendrimer coverage ratio of 97:1 was confirmed by elemental analysis of the carbon and sulfur content of the conjugate. Similar loading values were obtained by 1H NMR analysis of the aromatic and alkyl protons, however, the reproducibility of loading values determined by this method was poorer than SEC and combustion analysis. The conjugation reaction was performed in H2O, DMSO and mixtures of the two, the extent of ligand/dendrimer ratio was found to be unaffected by the choice of solvent. The phosphonate esters of the conjugate 10 were finally removed under identical conditions to those used in the preparation of the ligand 2, HBr and AcOH, to afford the conjugate 11.
Synthesis of the dendrimer-based pH responsive contrast agent Gd11. i) H2/Pd on C/H2O; ii) SCCl2/CHCl3/H2O pH 2; iii) G-5 PAMAM dendrimer/H2O pH 8; iv) 30% HBr in AcOH.
Formation of the gadolinium complexes of 2
also requires special attention. With most DOTA–tetraamide ligands, this step is relatively simple; however, we have recently reported that the phosphonate groups of 1
can interfere with these complexation reactions[12
] so care was taken to perform the complexation reactions of both 2
at pH 9 in order to ensure that the gadolinium ion was bound by the macrocyclic coordination cage. In the case of ligand 2
equimolar amounts of ligand and gadolinium chloride hexahydrate were reacted together at pH 9 and 60 °C in aqueous solution. No further purification was undertaken. However, 1.2 equivalents of gadolinium chloride were used in the reaction with 11
to ensure complete reaction of the ligands. After 48 h at 40 °C and pH 9 in aqueous solution the excess gadolinium was removed by addition of EDTA followed by dialysis in water (12 kD molecular weight cut-off, Sigma Aldrich). The conjugate Gd11
was then further purified by diafiltration with Centricon C-10 (10 kD cut-off) in water at pH 7.4. Although addition of the Gd3+
ion into each ligand of the conjugate would lead to a substantial increase in molecular weight, this increase would be expect to have little or no effect upon the hydrodynamic volume of the conjugate. Thus, size exclusion HPLC was used to verify that the apparent molecular weight remained near 140 kD.
The relaxivity pH profile of Gd2 was recorded and compared with that of Gd1 to ensure that the introduction of the benzylic function on the macrocyclic ring did not negatively impact the pH responsive properties of the complex. The relaxivity pH profiles of Gd1 and Gd2 () are comparable, rising and falling to approximately the same relaxivity at approximately the same pH. The only slight difference between the two profiles is that the relaxivity of Gd2 is slightly higher than that of Gd1 between pH 4 and 6. This may be a reflection of slight changes in the protonation constants of the phosphonates, but nonetheless indicates that introduction of the nitrobenzyl substituent does not negatively impact the behaviour of Gd2. Like Gd1 the relaxivity of Gd2 changes over a physiologically relevant pH range. The relaxivity pH profile of Gd11 is also shown (); here the advantage of conjugating the low molecular weight chelate to a dendrimer is immediately apparent. The relaxivity of Gd11 changes over the same physiologically relevant pH range as that of Gd1 and Gd2 but is much higher on a per Gd3+ basis, rising from 10.8 mm−1 s−1 at pH 9.5 to 24.0 mm−1 s−1 at pH 6. (On a per molecule basis relaxivity rises from 1037 mm−1 s−1 to 2304 mm−1 s−1.) This equates to a relaxivity pH response, Δr1, of 122% on passing from pH 9.5 to 6.0, more than doubling the Δr1 of Gd1 and Gd2, Δr1=51 and 59%, respectively, over the same pH range. Although the pH profiles of Gd1 and Gd2 exhibit a drop in relaxivity on passing below pH 6 the profile of Gd11 cannot be measured below this pH since the dendrimer conjugate precipitates from solution immediately below pH 5.9. This is presumably the result of the high molecular weight conjugate suddenly reaching its isoelectric point.
Figure 2 Relaxivity pH profiles of Gd1 () Gd2 (○) and Gd11 (●) recorded at 20 MHz and 298 K. Relaxivity is expressed per Gd3+ ion.
The difference in relaxivity between the “on” (pH 6) and “off” (pH 9.6) states of the dendrimer-based pH responsive agent was improved by more than a factor of 2 by slowing molecular rotation. This should render the dendrimer-conjugate, Gd11, a more effective pH responsive agent for imaging tissue pH. In order to assess the origins of this improvement in pH responsive behaviour, nuclear magnetic resonance dispersion (NMRD) profiles of Gd11 were recorded at high and low pH values (). At both pH 6.5 and at pH 9.3, the relaxivity increased at lower temperatures indicating that the observed relaxivities are not limited by slow water proton exchange between complex and bulk water. Comparing the high field (1–100 MHz) regions of NMRD profiles recorded at the same temperature we can see that at higher pH (9.3) the curve is flatter and has a lower magnitude than at lower pH (6.5). This indicates that at pH 9.3 the effective correlation time, τC, which is responsible for modulating relaxation, is shorter than it is at pH 6.5.
Figure 3 NMRD profiles of Gd11 recorded at pH 6.5 (top) and pH 9.3 (bottom); : 5, : 15, ●: 25, ○: 35°C.
Owing to the large number of ionizable phosphonate and dendrimer amine groups on the dendrimer, multiple protonated species must exist at each pH value. This complicates any attempt to “fit” these NMRD profiles in a quantitative, meaningful way. Nonetheless, it is useful examine the parameters that influence relaxivity in a qualitative sense in order to probe which factors are responsible for the observed behaviour. One may assume that each of the 96 Gd3+
chelates on the dendrimer surface has one water molecule in its inner coordination sphere. In addition, the phosphonate groups of each pendant arm may form hydrogen-bonding interactions with nearby water molecules forming a second hydration sphere. The extent of this second hydration sphere is likely to vary as the phosphonates are protonated or deprotonated with changing pH. Over a certain pH range it has also been shown that these phosphonates can catalyse exchange of protons from the coordinated water molecule to the bulk solvent.[12
] Finally there is an outer-sphere contribution to relaxivity resulting from the diffusion water molecules of the bulk solvent close by the slowly tumbling Gd3+
chelates. This outer-sphere effect depends primarily on the rate of diffusion of water and is insensitive to changes in pH.
complexes to dendrimers is a common strategy for slowing the rotational dynamics of paramagnetic complexes.[30
] In order to quantitatively describe the rotational dynamics of these dendrimer systems, the Lipari–Szabo approach is typically used.[38
] This model employs two correlation times; a long correlation time that defines the global motion of the entire dendrimer conjugate, τg
, and a second shorter correlation time, τl
, that reflects the local motion of the metal complex about its point of attachment to the dendrimer. This fast local motion is superimposed upon the slower global motion of the dendrimer. For systems conjugated to G5-PAMAM dendrimers the global correlation time, τg
, has been reported on the order of 4–5 ns.[42
] The correlation time describing the local motion, τl
, of the complex range from 0.07–0.76 ns.[41
] The relative weighting of these two correlation times is given by an order parameter, S2
, that can range from 0 (where local motion is dominant) to 1 (where isotropic, global motion is dominant). For dendrimers modified with Gd3+
was found to range from 0.28–0.5.
We assumed that similar rotational dynamics applied to Gd11
and these parameters to simulate the high field region (1–100 MHz) of an NMRD profile (). Electron-spin relaxation parameters were fixed at values similar to those used elsewhere.[40
] When the rate of proton exchange between water molecules associated with the complex (either 1st or 2nd hydration sphere) and the bulk solvent is extremely fast (short τM
) the high field NMRD profile is flat and relaxivity low (). This is because these protons have a low probability of being relaxed before they exchange back into the bulk solvent. At the other extreme, very slow exchange that is, very long τM
, the profile is also flat and relaxivity low () because these protons, once relaxed by Gd3+
, remain on the complex preventing others from being relaxed. So in both cases relaxation is not effectively transferred to the bulk solvent and the T1
of the bulk remains long. Between these two extremes, where 1 ns < τM
< 1000 ns, the profile is characterized by higher relaxivities and a “hump” between 10 and 60 MHz (). The magnitude of the relaxivity in the profiles will depend on these exchange kinetics, but also on the number of exchangeable protons and their distance from Gd3+
. However, these latter two parameters will not affect the shape of the NMRD profile.
Figure 4 Effect of proton residence lifetime (τM) on the relaxivity of Gd3+-G5 PAMAM dendrimer conjugates in the fast (top) and slow (bottom) exchange regimes. The simulated profiles are plotted at same scale using parameters: τg=4.5 ns, τ (more ...)
In order to mimic the flat, field independent behaviour observed for Gd11
at pH 9.3, the proton residence lifetime, τM
, must be either very short (<1 ns) or very long (>1 µs). The rate of water proton exchange is temperature dependent; as the temperature is lowered τM
becomes longer. This increase in τM
applies to both water molecules in the inner and second hydration spheres; however, it has an opposing effect on the relaxivity of each. The water protons of the inner-sphere water molecule of Gd1
were found to be in slow exchange with the bulk,[12
] and so these protons contribute poorly to the overall relaxivity. In contrast, water protons of a second hydration sphere are known to undergo very rapid exchange that also limits their contribution to relaxivity.[22
] Whereas making τM
of the inner sphere longer (lower temperature) will not result in an increase in relaxivity, making τM
of the second sphere longer could bring these protons into a range where they are able to contribute more substantially to relaxivity (). Inspecting the NMRD profile of Gd11
recorded at pH 9.3 and 35 °C, the high field region is flat suggesting that exchange of inner-sphere water protons is too slow and exchange of second sphere water protons too fast for a relaxivity enhancement “hump” to be observed at high field. As the temperature is taken down to 5 °C the profile begins to take on the appearance of a small “hump” at high field. Clearly exchange from the inner-sphere water protons will continue to be too slow to provide a contribution to relaxivity at lower temperatures. The small increase in relaxivity must therefore be the result of slowing the exchange rate of second-sphere water protons into a range that allows some contribution to relaxivity. This second-sphere contribution to relaxivity is not observed for either Gd1
because their rotational dynamics are too rapid. Given the small size of the high field relaxivity hump, exchange of second-sphere water molecules is likely to be in the range of τM
Over a certain pH range, the phosphonate groups of the pendant arms of the low molecular weight complexes Gd1
, catalyse exchange of inner-sphere water protons with the bulk solvent.[12
] The fast rotational dynamics (τg
≈0.1 ns) of these low molecular weight chelates limits their relaxivity but conjugation to the dendrimer lifts this restriction in Gd11
and so the observed relaxivity is higher by a factor of almost 5. At pH 6.5 and 35 °C the NMRD profile of Gd11
already has a slight high field “hump” which becomes more pronounced as the temperature is lowered. From studies on Gd1
, a contribution to this high field relaxivity from the inner-sphere proton exchange is expected. However, a contribution from protons in the second hydration sphere is also apparent. This is most clearly seen when the temperature is lowered. The τM
of inner-sphere water protons of Gd1
was found to be on the order of microseconds[12
] and so as the temperature is lowered the inner-sphere relaxivity should decrease as exchange becomes increasingly limited (cf. ). The observed relaxivity increases with decreasing temperature indicating that a substantial second-sphere component must be present (). The fact that the increase in high field relaxivity with decreasing temperature is larger at pH 6.5 than it is at pH 9.3 suggests that either the second hydration sphere is larger or it is more ordered, leading to longer τM
values, at pH 6.3 than at 9.3. Gd11
would seem to be a rare example of a system in which relaxivity is limited both by prototropic exchange in the 2nd-sphere that is too fast and by water exchange in the inner-sphere that is too slow.
It is worth noting that a third factor may also play a role in improving the relaxivity pH response of Gd11
complexes that exhibit no pH response have been found to behave as pH responsive agents once conjugated to PAMAM dendrimers.[44
] The origin of this phenomenon is thought to be changes in the internal motion of the dendrimer itself as the pH changes. Protonation of amines within the body of the dendrimer is believed to make the dendrimer more rigid making τR
longer at lower pH. Thus the relaxivity of agents conjugated to these dendrimers has been found to increase as the pH drops. It is likely that, in addition to the interplay of inner-sphere and second-sphere water proton exchange rates, a third contribution to the pH responsive behaviour of Gd11
arises from changes in the rigidity of the dendrimer with changes in pH.