Patent Application
20150297761
1. Field of the Invention
The invention relates to contrast agents with high relaxivity for MRI scanners that are operated at higher magnetic fields. The invention also relates to methods for preparing the contrast agents.
2. Description of the Related Art
When a substance such as human tissue is subjected to a uniform magnetic field (polarizing field B0), the individual magnetic moments of the excited nuclei in the tissue attempt to align with this polarizing field, but precess about it in random order at their characteristic Larmor frequency. If the substance, or tissue, is subjected to a magnetic field (excitation field B1) that is in the x-y plane and that is near the Larmor frequency, the net aligned moment, Mz, may be rotated, or “tipped”, into the x-y plane to produce a net transverse magnetic moment Mt. A signal is emitted by the excited nuclei or “spins”, after the excitation signal B1 is terminated, and this signal may be received and processed to form an image.
In magnetic resonance imaging (MRI) systems, the excited spins induce an oscillating sine wave signal in a receiving coil. The frequency of this signal is near the Larmor frequency, and its initial amplitude, is determined by the magnitude of the transverse magnetic moment Mt. The amplitude, A, of the emitted NMR signal decays in an exponential fashion with time, t. The decay constant 1/T*2 depends on the homogeneity of the magnetic field and on T2, which is referred to as the “spin-spin relaxation” constant, or the “transverse relaxation” constant. The T2 constant is inversely proportional to the exponential rate at which the aligned precession of the spins would dephase after removal of the excitation signal B1 in a perfectly homogeneous field. The practical value of the T2 constant is that tissues have different T2 values and this can be exploited as a means of enhancing the contrast between such tissues.
Another important factor that contributes to the amplitude A of the NMR signal is referred to as the spin-lattice relaxation process that is characterized by the time constant T1. It describes the recovery of the net magnetic moment M to its equilibrium value along the axis of magnetic polarization (z). T2 relaxation is associated with a decrease in spin coherence, and T1 relaxation occurs due to a paramagnetic shift at the probe site and subsequent exchange of bound protons with the surrounding bulk water. The T1 time constant is longer than T2, much longer in most substances of medical interest. As with the T2 constant, the difference in T1 between tissues can be exploited to provide image contrast.
The reciprocals of these relaxation time constants are termed relaxation rates and denoted R1 and R2 where R1=1/T1 and R2=1/T2.
Contrast agents are exogenous molecules or materials that can alter the relaxation properties of tissue and induce image contrast. Contrast agents are typically paramagnetic, superparamagnetic, or ferromagnetic materials. Contrast agents are also sometimes referred to as imaging probes.
The extent to which a given contrast agent can alter the relaxation rate is termed relaxivity. Relaxivity is defined as the difference in the relaxation rate of a sample measured with and without the contrast agent. This relaxation rate difference is then normalized to the concentration of the contrast agent. Relaxivity is expressed as a lowercase “r” with a subscript “1” or “2” which refers to either the longitudinal or transverse relaxivity respectively. For instance longitudinal relaxivity, r1, is defined as r1=(R1−R10)/C where R1 is the relaxation rate in s−1 measured in the presence of the contrast agent, R10 is the relaxation rate in s−1 measured in the absence of contrast agent, and C is the concentration in mM of the contrast agent. Relaxivity has units of mM−1s−1. For contrast agents that contain more than one metal ion, relaxivity can be expressed in terms of the metal ion concentration (‘per ion’ or ‘ionic relaxivity’) or in terms of the molecular concentration (‘per molecule’ or ‘molecular relaxivity’). Relaxivity is an inherent property of contrast agents.
In an effort to elicit clinically-desired contrasts, MRI contrast agents have been developed that are designed to affect the relaxation periods. Not surprisingly, there are contrast agents that are used clinically to adjust T1 contrast and those that are used clinically to adjust T2 contrast.
T1-weighted (T1w) imaging provides image contrast where tissues or regions of the image are bright (increased signal intensity) when the T1 of water in that region is short. One way to increase image contrast is to administer a paramagnetic complex or material based on gadolinium (Gd), manganese, or iron. For example, clinically utilized Gd(III) imaging probes comprise nonacoordinate, ternary complexes where the Gd(III) ion is held within an octadentate polyaminocarboxylate ligand and coordinated by one rapidly exchanging water ligand. Due to strong paramagnetism (S=7/2) and slow electronic relaxation (T1e), complexes of Gd(III) are ideal for generating T1 contrast (example 5, ref 1). This paramagnetic contrast agent shortens the T1 of water molecules that it encounters and results in positive image contrast. The degree to which a given concentration of contrast agent can change T1 is the relaxivity as noted above. Compounds that have higher relaxivity provide greater T1w signal enhancement than compounds with low relaxivity; alternatively a high relaxivity compound can provide equivalent signal enhancement to a low relaxivity compound but at a lower concentration than the low relaxivity compound. Thus, high relaxivity compounds are desirable because they enable greater enhancement of lesions and improve diagnostic confidence; alternately, they can be used at lower doses and thus improve the safety margin of the contrast agent.
An increasing number of MRI scanners being sold today operate at higher magnetic fields, e.g., at 3 Tesla or 7 Tesla. The signal:noise ratio (SNR) increases with increasing magnetic field. In addition, the inherent T1 of tissue is also decreasing with increasing field and thus the sensitivity of Gd-based contrast agents should increase with field if probe relaxivity (r1) is field independent. Unfortunately for many contrast agents, r1 typically decreases with field faster than the decrease in baseline T1. However, by controlling the rotational dynamics of the contrast agent, it is possible to create high relaxivity contrast agents that exhibit high relaxivity at high fields.
To optimize relaxivity, the contrast agent should undergo rotational diffusion with a rate close to the Larmor frequency of hydrogen at the applied magnetic field, e.g., ˜127 MHz at 3 Tesla. In general, there are three modular parameters readily available to the chemist: the rate of inner-sphere water exchange (kex=1/τm; τm=mean residency time of the H2O ligand), the rotational correlation time of the complex (τR) and the hydration number of the Gd(III) ion (q). The property τm is dictated by the ligand frame and choice of donor groups, and the manifestations of commonly used functional groups on τm have been explored in detail (example 5, refs 4, 5). The optimal range of τm values depends on both τR and magnetic field, (example 5, refs 5, 7) however 10<τm<30 ns is optimal across all field strengths. Increasing τR can afford tremendous r1 enhancement at relatively low field strength (<1.5 T) (example 5, ref 6). This strategy has met with much success by either multimerization (example 5, ref 8) or through covalent or non-covalent conjugation to macromolecular entities (example 5, refs 9-11). In order to design molecules with precisely tuned dynamics it is desirable to have the paramagnetic ion rotate isotropically along with the entire molecule. For larger molecules, the metal ion complex may be tethered to another part of the molecule via a flexible linker. This flexible linker results in local rotational motion that is faster than the overall rotational diffusion of the entire molecule. Fast local motion limits relaxivity.
Alternatively, modulation of the hydration number q can significantly affect the r1 of contrast agents including Gd(III) complexes, and r1 tends to scale with this parameter independent of field. However, an increase in q requires a reduction in available ligand donors and can come at the cost of reduced thermodynamic stability and kinetic inertness with respect to transchelation of the Gd(III) ion. Therefore, one must be judicious in ligand design. A handful of q>1 Gd(III) complexes featuring hexa- and heptadentate ligands have been prepared and evaluated, (example 5, refs 12-16) with notable examples highlighted in
Therefore, there is a need for higher relaxivity contrast agents for magnetic resonance imaging systems being operated at higher magnetic fields. There is also a need for methods for preparing such higher relaxivity MRI contrast agents.
The invention meets the foregoing needs by providing a method of increasing the relaxivity of a contrast agent having a metal ion complexed to a chelator. By tethering the metal ion complex to the remainder of the molecule by at least two points of attachment, local motion is limited and higher relaxivity can be achieved.
In one non-limiting example version of the invention, we investigated applications of the alanine analogue of Gd(DOTA), Gd(DOTAla), wherein Gd is gadolinium and DOTA is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid. Fmoc (fluorenylmethyloxycarbonyl) protected DOTAla suitable for solid phase peptide synthesis was synthesized from cyclen and a mesylated serine derivative in five steps and 15% over all yield. The integration of this unnatural amino acid into polypeptide structures followed by complexation with Gd(III) results in very rigid attachment of the metal ion complex to the peptide backbone. Rigid molecular structures provide fewer degrees of rotational freedom, resulting in greater control over the rotational dynamics and resultant relaxivity. In the case of Gd(DOTAla), the metal ion complex is tethered to the peptide via the amino acid side chain to the DOTA moiety and via a dative bond from an amide oxygen to the Gd(III) ion.
We also investigated the application of Gd(DOTAla) as a blood pool imaging agent, by targeting human serum albumin (HSA). We took advantage of the possibility to attach multiple functional groups capable of targeting HSA. As the system is modular, rapid synthesis of a variety of derivatives is possible and allows for accelerated screening for the optimal contrast agent. We have also prepared multimeric peptide based contrast agents of defined size and relaxivity. These peptide based contrast agents have the general synthetic flexibility of peptides and can also incorporate other imaging reporters (e.g. fluorescent moieties or positron emitters) or specific targeting vectors.
We provide a synthetic intermediate which allows for efficient multimerization or integration of the chelate DOTAla into polypeptides using solid phase peptide synthesis. We have also found the corresponding Gd complexes to have excellent properties for development of molecular imaging agents for MRI. The modular nature of the invention allows for facile synthesis of multimodal imaging probes, targeted imaging probes, or theranostics. Furthermore, the well-defined structure of (DOTAla) enables rational peptide or protein design. Other paramagnetic metals may be incorporated that provide relaxation or chemical shift effects that can be used for structural elucidation of proteins.
Furthermore, we have designed and evaluated a new ligand framework, CyPic3A, capable of forming ternary complexes with Gd(III) featuring two coordinated waters. The two bound waters afford favorable r1 and appear to be impervious to displacement by endogenously encountered bidentate ions such as phosphate, carbonate and lactate. Despite the heptadentate nature of the ligand, CyPic3A forms Gd(III) complexes with thermodyanamic stability and kinetic inertness comparable to FDA approved probes employing octadenate ligands. CyPic3A holds promise regarding the development of highly efficient imaging probes amenable to use across a wide range of magnetic fields.
These and other features, aspects, and advantages of the present invention will become better understood upon consideration of the following detailed description, drawings, and appended claims.
The invention provides a compound for diagnosing or treating a subject. In one preferred embodiment of the invention, the compound is used as a contrast agent in a diagnostic imaging technique such as magnetic resonance imaging (MRI), positron emission tomography (PET), and/or single-photon emission computed tomography (SPECT). For purposes of the present invention, “treating” or “treatment” describes the management and care of a patient for the purpose of combating a disease, condition, or disorder. The terms embrace both preventative, i.e., prophylactic, and palliative treatment. Treating includes the administration of a compound of the present invention to prevent the onset of the symptoms or complications, alleviating the symptoms or complications, or eliminating the disease, condition, or disorder. A “subject” is a mammal, preferably a human.
The form in which the compound is administered to the subject is not critical. For example, the compound of the invention can be administered directly to tissue being diagnostically imaged or treated, to a body fluid that contacts the tissue, or to a body location from which the compound can diffuse or be transported to the tissue being diagnostically imaged or treated.
The compound can be administered alone or as part of a pharmaceutically acceptable composition. The relative amounts of the compound of the invention, a pharmaceutically acceptable carrier, and any additional active ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the human treated and further depending upon the route by which the compound is to be administered. Other pharmaceutically active compounds can be selected to treat the same disease as a compound of the invention or a different disease. A compound of the invention, optionally comprising other pharmaceutically active compounds, can be administered to a subject parenterally, for example, intravenously, intramuscularly, subcutaneously, intracerebrally or intrathecally.
In one non-limiting example embodiment of the invention, the compound for diagnosing or treating a subject has the formula (I):
wherein A is a first amino acid residue, and B is a chelate complex comprising a chelator and a metal ion, the chelator comprising a ring of atoms. The chelator forms at least one coordinate bond with the metal ion, and the first amino acid residue is bonded to an atom of the ring of the chelator. The first amino acid residue has a carbonyl group oxygen that forms a coordinate bond with the metal ion. R1 is a moiety comprising hydrogen, or an amino acid residue, or combinations thereof; and R2 is a moiety comprising hydrogen, or an amino acid residue, or combinations thereof. At least one of R1 and R2 comprises an amino acid residue. By “amino acid residue”, we mean an amino acid that has a hydrogen ion removed from the amine end, or a hydrogen ion or hydroxyl ion removed from the carboxyl end, or both.
The first amino acid residue can be selected from residues of alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, pyrrolysine, proline, glutamine, arginine, serine, threonine, selenocysteine, valine, tryptophan, and tyrosine. In non-limiting example versions of the compound, the peptide backbone (R1 and A and R2) can include two or more residues such as residues of alanine, cysteine, phenylalanine, glycine, and tyrosine. In another non-limiting example, R1 includes a cysteine residue, and R2 includes a cysteine residue. Optionally, the cysteine residues in R1 and R2 can be linked by a disulfide bond. Preferably, the first amino acid residue is an alanine residue.
The peptide backbone can include any number of residues; however, for ease of synthesis and reproducibility in clinical trials, it may be preferred to limit the residues in the peptide to 20 or less, and more preferably, 10 or less. The peptide backbone can be attached to pharmacologically active groups, immunoreactive haptens, polymers, nanoparticles, proteins, other peptides, enzymes, drugs, and vitamins. In one example form, the peptide backbone is attached to a protein, enzyme, peptide, antibody, or drug that can target a specific site (e.g., tumor) in a subject (human or animal) undergoing a diagnostic medical procedure. For example, at least one of R1 and R2 can comprise a blood plasma binding moiety, or at least one of R1 and R2 can comprise a targeting moiety that can target a site in the subject.
In another non-limiting example embodiment of the invention, the compound for diagnosing or treating a subject has the formula (II):
wherein A, R1, and R2 are as defined above, and at least one of R1 and R2 comprises an amino acid residue. R3, R4, and R5 can be independently selected from the group consisting of H, CH2CO2H, CH2CH2CO2H, CH2C(O)NR6R7, CH2NHCOR6, CH2C(O)N(OH)R6, CH2C(O)NHSO2R6, CH2NHSO2R6, CH2N(OH)C(O)R6, CH2P(R6)O2R7, CH2PO3R6R7, wherein R6 and R7 are independently selected from the group consisting of H, CO2H, C1-C6 alkyl, C1-6CO2H, CH(CO2H)C1-6CO2H, C1-6CF3, C1-6CCl3, C1-6CBr3, C1-6Cl3, or C1-6PO3R9R10, wherein R9 and R10 are independently selected from the group consisting of H, CO2H, C1-C6 alkyl, C1-6CO2H, CH(CO2H)C1-6CO2H. M is a metal ion, and an atom of at least one of R3, R4, and R5 in the compound forms a coordinate bond with the metal ion.
In one non-limiting preferred version of the invention, the compound has the formula (III):
wherein R1 and R2 are as defined above, and at least one of R1 and R2 comprises an amino acid residue, and M is a metal ion, preferably Gd3+. In formula (III), group A of the compounds of Formulas (I) and (II) is alanine.
Compounds according to the invention can also synthesized to have a multimeric structure. An example multimeric compound for diagnosing or treating a subject has the formula (IV):
wherein A is a first amino acid residue, and B is a chelate complex comprising a chelator and a metal ion wherein the chelator comprises a ring of atoms. The chelator forms at least one coordinate bond with the metal ion, and the first amino acid residue is bonded to an atom of the ring of the chelator. The first amino acid residue has a carbonyl group oxygen that forms a coordinate bond with the metal ion. R11 is a moiety comprising hydrogen, or an amino acid residue, or combinations thereof; R12 is nothing or a moiety comprising hydrogen, or an amino acid residue, or combinations thereof, and R13 is a moiety comprising hydrogen, or an amino acid residue, or combinations thereof. At least one of R11 and R13 comprises an amino acid residue, and n is an integer of 2 or more.
In the compound of formula (IV),
can have the formula (V)
wherein A, R1, and R2 are as defined above, and at least one of R1 and R2 comprises an amino acid residue. R3, R4, and R5 can be independently selected from the group consisting of H, CH2CO2H, CH2CH2CO2H, CH2C(O)NR6R7, CH2NHCOR6, CH2C(O)N(OH)R6, CH2C(O)NHSO2R6, CH2NHSO2R6, CH2N(OH)C(O)R6, CH2P(R6)O2R7, CH2PO3R6R7, wherein R6 and R7 are independently selected from the group consisting of H, CO2H, C1-C6 alkyl, C1-6CO2H, CH(CO2H)C1-6CO2H, C1-6CF3, C1-6CCl3, C1-6CBr3, C1-6Cl3, or C1-6PO3R9R10, wherein R9 and R10 are independently selected from the group consisting of H, CO2H, C1-C6 alkyl, C1-6CO2H, CH(CO2H)C1-6CO2H. M is a metal ion, and an atom of at least one of R3, R4, and R5 in the compound forms a coordinate bond with the metal ion.
In the compound of formula (IV),
can have the formula (VI)
wherein M is the metal ion.
Group A in the compounds of Formulas (I), (II), and (IV) has limited rotational freedom with respect to group B due to very rigid attachment of the metal ion complex to the peptide backbone. Rigid molecular structures provide fewer degrees of rotational freedom, resulting in greater control over the rotational dynamics and resultant relaxivity. In the case of the compounds of Formulas (I) and (II) and (IV), the metal ion complex is tethered to the peptide via the amino acid (e.g., alanine) residue side chain to the chelator (e.g., DOTA) moiety and via a coordinate bond from an amide oxygen to the metal ion (e.g., Gd3+).
In the compounds of formulas (I) and (II) and (IV), the metal ion can be selected from ions of gadolinium, europium, terbium, manganese, iron, 45Ti, 51Mn, 52Mn, 52mMn, 52Fe, 60Cu, 61Cu, 62Cu, 64Cu, 67Cu, 67Ga, 68Ga, 72As, 86Y, 89Zr, 90Nb, 94mTc, 99mTc, 110In, 111In, 113In, 177Lu, 201Tl, 212Pb 213Bi, or 225Ac. The metal ion can be paramagnetic. The metal ion can be selected from paramagnetic metal ions having atomic numbers 21-29, 43, 44, and 57-83. The chelated metal ion enables the compound to be quantified by magnetic resonance imaging (MRI), positron emission tomography (PET), and/or single-photon emission computed tomography (SPECT). For MRI, the metal ion can be, for example, a gadolinium ion (Gd3+) or a manganese ion (Mn2+). For PET detection, the metal ion can be a positron-emitting radionuclide (e.g., 64Cu, 68Ga) which will annihilate to form two gamma rays which will be detected by the PET camera. For SPECT detection, the chosen metal ion (e.g., ions of 111In) should produce a large number of photons. Alternatively, a non-metal, non-chelated positron-emitting radionuclide (e.g., 18F, 11C, 13N, 15O, 75Br, 76Br, 82Rb, 124I) can replace an atom of the compound for PET detection.
In the compounds of formulas (I) and (II) and (III), at least one of R1 and R2 can comprise a fluorescent moiety. In the compounds of formula (IV), at least one of R11 and R13 can comprise a fluorescent moiety. Fluorescent compounds can be used in molecular imaging both in vitro and in vivo. For in vivo imaging, near infrared (NIR) fluorophores have ideal absorption/emission wavelengths between 550 and 1000 nanometers, which minimize autofluorescence interference from tissue and have minimal overlap with biological chromophores such as hemoglobin. By including an NIR fluorophore in any of the compounds of Formulas (I) and (II) and (III) and (IV), the compound can be successfully applied to in vivo imaging of tissue (such as a tumor). Preferably, the fluorescent moiety has an absorption wavelength maxima in the range of 650 to 850 nanometers for imaging of tissue. The fluorescent moiety can be selected from cyanine dyes, carbocyanine dyes, and CyAL dyes, such as the carbocyanine dyes described in United States Patent Application Publication No. 2011/0286933. In such an imaging method, the compound including an NIR fluorophore is administered to a region of interest of a subject, light is directed into the subject, fluorescent light emitted from the subject is detected, and the detected light is processed to provide an image that corresponds to the region of interest of the subject.
The compounds of Formulas (I) and (II) and (III) and (IV) can be advantageous when used as a contrast agent in MRI, particularly as a higher relaxivity contrast agent for magnetic resonance imaging systems being operated at higher magnetic fields. The compounds of Formulas (I), (II), (III) and (IV) can be synthesized to have a per-metal r1 relaxivity of greater than 4 mM−1s−1, preferably greater than 5 mM−1s−1, preferably greater than 6 mM−1s−1, preferably greater than 7 mM−1s−1, preferably greater than 8 mM−1s−1, preferably greater than 9 mM−1s−1, preferably greater than 10 mM−1s−1, or preferably greater than 11 mM−1s−1. When the compounds of Formulas (I) and (II) and (III) are synthesized such that least one of R1 and R2 comprises a blood plasma binding moiety, such as an albumin binding moiety, or when the compounds of formula (IV) are synthesized such that least one of R11 and R13 comprises a blood plasma binding moiety, such as an albumin binding moiety, the compounds can have a per-metal r1 relaxivity of greater than 5 mM−1s−1, or preferably greater than 10 mM−1s−1, or preferably greater than 15 mM−1s−1, or preferably greater than 20 mM−1s−1, or preferably greater than 25 mM−1s−1, or preferably greater than 30 mM−1s−1. Also, the compounds of Formulas (I) and (II) and (III) and (IV) can be synthesized to have a mean water residency time (τM) at 37° C. of 1 to 50 nanoseconds, more preferably 1 to 30 nanoseconds, more preferably 10 to 25 nanoseconds, or most preferably 15 to 20 nanoseconds.
Certain compounds are useful for synthesizing the compounds of Formulas (I) and (II). One particularly useful compound for synthesizing the compounds of Formulas (I) and (II), has the formula (VII):
wherein A is a first amino acid residue, and Z is a chelator comprising a ring of atoms. The first amino acid residue is bonded to an atom of the ring of the chelator, and the first amino acid residue has a carbonyl group oxygen that can form a coordinate bond with a metal ion. R1 is a moiety comprising hydrogen, or an amino acid residue, or combinations thereof, and R2 is a moiety comprising hydrogen, or an amino acid residue, or combinations thereof. At least one of R1 and R2 comprises an amino acid residue, and at least one of R1 and R2 comprises a fluorenylmethyloxycarbonyl moiety.
The invention provides a method of increasing the r1 relaxivity of a contrast agent. The contrast agent includes metal ion complexed to a chelator comprising a ring of atoms wherein the chelator forms at least one coordinate bond with the metal ion. In the method, a peptide scaffold is attached to the chelator such that the peptide scaffold has limited rotational freedom with respect to the chelator. The peptide scaffold includes a first amino acid residue, and the first amino acid residue is bonded to an atom of the ring of the chelator. The first amino acid residue also has a carbonyl group oxygen that forms a coordinate bond with the metal ion. In one non-limiting version, the chelator is 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid, the metal ion is Gd3+, and the first amino acid residue is alanine.
The invention provides a method for in vivo imaging of a subject. In the method, any of the compounds of Formulas (I) and (II) and (III) and (IV) are administered to the subject. One waits a time sufficient to allow the compound to accumulate at a tissue or cell site to be imaged, and the cells or tissues are imaged with a non-invasive imaging technique whose resolution is enhanced by the presence of the compound on or within the cells. The non-invasive imaging technique can be magnetic resonance imaging.
The invention provides a method of imaging a subject having been administered a dose of a contrast agent including any of the compounds of Formulas (I) and (II) and (III) and (IV). In the method, the subject is positioned in a magnet system configured to generate a polarizing magnetic field about at least a portion of a subject. A plurality of gradient coils configured to apply a gradient field to the polarizing magnetic field are energized. A radio frequency (RF) system configured to apply an excitation field to the subject and acquire magnetic resonance (MR) image data therefrom is controlled, and an image of the subject is reconstructed from the MR image data. Molecules in the subject that are subjected to the contrast agent have a modified one of a longitudinal relaxation period and a transverse relaxation period that is reflected in the image.
When any of the compounds of Formulas (I) and (II) and (III) and (IV) includes a molecular targeting group, other bioactive agents may be included in the compound. For example, a cytotoxic agent can be associated with any of the compounds of Formulas (I) and (II) and (III) and (IV). A cytotoxic agent is “associated” with a compound of the invention if the cytotoxic agent is directly or indirectly, physically or chemically bound to the compound. Non-limiting examples of chemical bonds include covalent bonds, ionic bonds, coordinate bonds, and hydrogen bonds. Indirect bonding can include the use of a group of atoms (i.e., a linker) that chemically links the cytotoxic agent and the compound. Non-limiting examples of physical bonding include physical adsorption and absorption. The cytotoxic agent can be a cytotoxin (e.g., ricin, pseudomonas exotoxin, diphtheria toxin). The cytotoxic agent can be a chemotherapeutic agent (e.g., alkylating agents, antagonists, plant alkaloids, intercalating antibiotics, enzyme inhibitors, antimetabolites, mitotic inhibitors, growth factor inhibitors, cell cycle inhibitors, enzymes, biological response modifiers). The cytotoxic agent can be a radiation-emitter (e.g., phosphorus-32, phosphorus-33, bromine-77, yttrium-88, yttrium-90, molybdenum-99m, technetium-99m, indium-111, indium-131, iodine-123, iodine-124, iodine-125, iodine-131, lutetium-177, rhenium-186, rhenium-188, bismuth-212, bismuth-213, astatine-211).
We have investigated other chelators and chelate complexes useful in the invention. These chelate complexes could be attached to amino acid residues using the methods of the invention.
In another non-limiting example embodiment of the invention, the compound has the formula (VIII):
wherein R16 is selected from substituted or unsubstituted alkyl carboxylates, substituted or unsubstituted cycloalkyl carboxylates, and substituted or unsubstituted heterocyclic carboxylates, wherein R17 is selected from substituted or unsubstituted alkyl carboxylates, substituted or unsubstituted cycloalkyl carboxylates, and substituted or unsubstituted heterocyclic carboxylates, and wherein R18 is selected from substituted or unsubstituted alkylenes and substituted or unsubstituted cycloalkylenes.
In one aspect, the compound of formula (VIII) is formulated such that R18 is further selected from unsubstituted cycloalkylenes, such as cyclohexylene. In another aspect, R16 is selected from unsubstituted alkyl carboxylates, such as C1-C20 alkyl carboxylate and in one case, carboxylate. In yet another aspect, R17 is a carboxyalkylpyridine, such as carboxy-(C1-C20)alkyl-pyridine and in one case, carboxymethylpyridine. In another example, the compound of formula (VIII) is formulated such that R16 is methyl carboxylate, R17 is carboxymethylpyridine, and R18 is cyclohexylene.
In another non-limiting example embodiment of the invention, the compound has the formula (IX):
wherein R16 is selected from substituted or unsubstituted alkyl carboxylates, substituted or unsubstituted cycloalkyl carboxylates, and substituted or unsubstituted heterocyclic carboxylates, wherein R17 is selected from substituted or unsubstituted alkyl carboxylates, substituted or unsubstituted cycloalkyl carboxylates, and substituted or unsubstituted heterocyclic carboxylates, wherein R18 is selected from substituted or unsubstituted alkylenes and substituted or unsubstituted cycloalkylenes, and wherein M is a metal ion.
In one aspect, the compound of formula (IX) is formulated such that R18 is selected from unsubstituted cycloalkylenes, such as cyclohexylene. In another aspect, R16 is selected from unsubstituted alkyl carboxylates, such as C1-C20 alkyl carboxylate, and in one case, methyl carboxylate. In yet another aspect, R17 is a carboxyalkylpyridine, such as carboxy-(C1-C20)alkyl-pyridine, and in one case, carboxymethylpyridine. In another example, the compound of formula (IX) is formulated such that R16 is methyl carboxylate, R17 is carboxymethylpyridine, and R18 is cyclohexylene.
The metal ion of formula (IX) may be selected based on a number of criteria. In one embodiment, the compound of formula (IX) is formulated such that the metal ion is selected from ions of gadolinium, europium, terbium, manganese, iron, 45Ti, 51Mn, 52Mn, 52mMn, 52Fe, 60Cu, 61Cu, 62Cu, 64Cu, 67Cu, 67Ga, 68Ga, 72As, 86Y, 89Zr, 90Nb, 94mTc, 99mTc, 110In, 111In, 113In, 177Lu, 201Tl, 212Pb 213Bi, or 225Ac. Alternatively, or in addition, the metal ion of formula (IX) is paramagnetic. In one aspect, the metal ion is selected from paramagnetic metal ions having atomic numbers 21-29, 43, 44, and 57-83.
In another aspect, the compound of formula (IX) is formulated to possess other desirable properties. For example, in one embodiment, the compound has a per-metal r1 relaxivity of greater than 4 mM−1s−1, preferably greater than 5 mM−1s−1, preferably greater than 6 mM−1s−1, preferably greater than 7 mM−1s−1, preferably greater than 8 mM−1s−1, preferably greater than 9 mM−1s−1, preferably greater than 10 mM−1s−1, preferably greater than 11 mM−1s−1. In another aspect, the compound of formula (IX) has a mean water residency time of 5 to 30 nanoseconds, preferably 10 to 25 nanoseconds, preferably 15 to 20 nanoseconds.
In yet another embodiment, the compound of formula (IX) is employed as a contrast agent for magnetic resonance imaging. In one aspect, the metal ion of formula (IX) is Gd3+. In another aspect, the compound is heptadentate and/or the metal ion coordinates with two molecules of water.
The invention further encompasses a number of methods. One non-limiting method relates to increasing the r1 relaxivity of a contrast agent having a metal ion complexed to a chelator including the step of attaching a peptide scaffold to the chelator such that the peptide scaffold has limited rotational freedom with respect to the chelator. In one aspect, the peptide scaffold includes a first amino acid residue that is bonded to an atom of the chelator. In another aspect, the first amino acid residue has a carbonyl group oxygen that forms a coordinate bond with the metal ion.
In one embodiment of the method, the chelator is CyPic3A as in
Yet another non-limiting method of the present invention for in vivo imaging of a subject includes the steps of (i) administering to the subject a compound such as the compound of formula (IX), (ii) waiting a time sufficient to allow the compound to accumulate at a tissue or cell site to be imaged; and (iii) imaging the cells or tissues with a non-invasive imaging technique whose resolution is enhanced by the presence of the compound on or within the cells. In one aspect, the non-invasive imaging technique is magnetic resonance imaging.
Still another non-limiting method relates to imaging a subject having been administered a dose of a contrast agent. The method includes the steps of (i) positioning the subject in a magnet system configured to generate a polarizing magnetic field about at least a portion of a subject; (ii) energizing a plurality of gradient coils configured to apply a gradient field to the polarizing magnetic field; (iii) controlling a radio frequency (RF) system configured to apply an excitation field to the subject and acquire magnetic resonance (MR) image data therefrom; and (iv) reconstructing an image of the subject from the MR image data. In one aspect, the contrast agent includes the compound of formula (IX). In another aspect, molecules in the subject that are subjected to the contrast agent have a modified one of a longitudinal relaxation period and a transverse relaxation period that is reflected in the image.
In addition to the compounds described above, another embodiment relates to compounds having the formula (VIII):
wherein R16 is selected from substituted or unsubstituted alkyl carboxylates, substituted or unsubstituted cycloalkyl carboxylates, substituted or unsubstituted heterocyclic carboxylates, and amino acids, wherein R17 is selected from substituted or unsubstituted alkyl carboxylates, substituted or unsubstituted cycloalkyl carboxylates, substituted or unsubstituted heterocyclic carboxylates, and amino acids and wherein R18 is selected from substituted or unsubstituted alkylenes and substituted or unsubstituted cycloalkylenes.
Yet another embodiment relates to compounds having the formula (IX):
wherein R16 is selected from substituted or unsubstituted alkyl carboxylates, substituted or unsubstituted cycloalkyl carboxylates, substituted or unsubstituted heterocyclic carboxylates, and amino acids, wherein R17 is selected from substituted or unsubstituted alkyl carboxylates, substituted or unsubstituted cycloalkyl carboxylates, substituted or unsubstituted heterocyclic carboxylates, and amino acids, wherein R18 is selected from substituted or unsubstituted alkylenes and substituted or unsubstituted cycloalkylenes, and wherein M is a metal ion.
In another non-limiting example embodiment of the invention, the compound has the formula (X):
wherein A is selected from
wherein R3, R4, and R5 are independently selected from the group consisting of H, CH2CO2H, CH2CH2CO2H, CH2C(O)NR6R7, CH2NHCOR6, CH2C(O)N(OH)R6, CH2C(O)NHSO2R6, CH2NHSO2R6, CH2N(OH)C(O)R6, CH2P(R6)O2R7, CH2PO3R6R7, wherein R6 and R7 are independently selected from the group consisting of H, CO2H, C1-C6 alkyl, C1-6CO2H, CH(CO2H)C1-6CO2H, C1-6CF3, C1-6CCl3, C1-6CBr3, C1-6Cl3, or C1-6PO3R9R10, wherein R9 and R10 are independently selected from the group consisting of H, CO2H, C1-C6 alkyl, C1-6CO2H, CH(CO2H)C1-6CO2H;
wherein R20 and R21 are independently selected from
and
wherein M a metal ion.
The metal ion of formula (X) can be selected from ions of gadolinium, europium, terbium, manganese, iron, 45Ti, 51Mn, 52Mn, 52mMn, 52Fe, 60Cu, 61Cu, 62Cu, 64Cu, 67Cu, 67Ga, 68Ga, 72As, 86Y, 89Zr, 90Nb, 94mTc, 99mTc, 110In, 111In, 113In, 177Lu, 201Tl, 212Pb 213Bi, or 225Ac. The metal ion can be paramagnetic. The metal ion can be selected from paramagnetic metal ions having atomic numbers 21-29, 43, 44, and 57-83. The compound of formula (X) can have a per-metal r1 relaxivity of greater than 3 mM−1s−1, preferably greater than 4 mM−1s−1, preferably greater than 5 mM−1s−1, preferably greater than 6 mM−1s−1. The compound of formula (X) can have a mean water residency time of 5 to 30 nanoseconds, preferably 5 to 20 nanoseconds, preferably 5 to 10 nanoseconds. The compound of formula (X) can be a contrast agent for magnetic resonance imaging. The metal ion of formula (X) can be Gd3+. In one structure of the compound of formula (X), R3 is CH2CO2H, R4 is CH2PO3R6R7, and R5 is CH2CO2H wherein R6 and R7 are H.
Yet another non-limiting method of the present invention for in vivo imaging of a subject includes the steps of (i) administering to the subject a compound such as the compound of formula (X), (ii) waiting a time sufficient to allow the compound to accumulate at a tissue or cell site to be imaged; and (iii) imaging the cells or tissues with a non-invasive imaging technique whose resolution is enhanced by the presence of the compound on or within the cells. In one aspect, the non-invasive imaging technique is magnetic resonance imaging.
Still another non-limiting method relates to imaging a subject having been administered a dose of a contrast agent. The method includes the steps of (i) positioning the subject in a magnet system configured to generate a polarizing magnetic field about at least a portion of a subject; (ii) energizing a plurality of gradient coils configured to apply a gradient field to the polarizing magnetic field; (iii) controlling a radio frequency (RF) system configured to apply an excitation field to the subject and acquire magnetic resonance (MR) image data therefrom; and (iv) reconstructing an image of the subject from the MR image data. In one aspect, the contrast agent includes the compound of formula (X). In another aspect, molecules in the subject that are subjected to the contrast agent have a modified one of a longitudinal relaxation period and a transverse relaxation period that is reflected in the image.
Referring to
The pulse sequence server 110 functions in response to instructions downloaded from the workstation 102 to operate a gradient system 118 and a radiofrequency (“RF”) system 120. Gradient waveforms necessary to perform the prescribed scan are produced and applied to the gradient system 118, which excites gradient coils in an assembly 122 to produce the magnetic field gradients Gx, Gy, and Gz used for position encoding MR signals. The gradient coil assembly 122 forms part of a magnet assembly 124 extending about a bore 125 formed there through and includes a polarizing magnet 126 and a whole-body RF coil 128.
RF excitation waveforms are applied to the RF coil 128, or a separate local coil (not shown), by the RF system 120 to perform the prescribed magnetic resonance pulse sequence. Responsive MR signals detected by the RF coil 128, or a separate local coil (not shown), are received by the RF system 120, amplified, demodulated, filtered, and digitized under direction of commands produced by the pulse sequence server 110. The RF system 120 includes an RF transmitter for producing a wide variety of RF pulses used in MR pulse sequences. The RF transmitter is responsive to the scan prescription and direction from the pulse sequence server 110 to produce RF pulses of the desired frequency, phase, and pulse amplitude waveform. The generated RF pulses may be applied to the whole body RF coil 128 or to one or more local coils or coil arrays.
The RF system 120 also includes one or more RF receiver channels. Each RF receiver channel includes an RF amplifier that amplifies the MR signal received by the coil 128 to which it is connected, and a detector that detects and digitizes the I and Q quadrature components of the received MR signal. The magnitude of the received MR signal may thus be determined at any sampled point by the square root of the sum of the squares of the I and Q components:
M=√{square root over (I2+Q2)} Eqn. (1);
and the phase of the received MR signal may also be determined:
The pulse sequence server 110 also optionally receives patient data from a physiological acquisition controller 130. The controller 130 receives signals from a number of different sensors connected to the patient, such as electrocardiograph (“ECG”) signals from electrodes, or respiratory signals from a bellows or other respiratory monitoring device. Such signals are typically used by the pulse sequence server 110 to synchronize, or “gate,” the performance of the scan with the subject's heart beat or respiration.
The digitized MR signal samples produced by the RF system 120 are received by the data acquisition server 112. The data acquisition server 112 operates in response to instructions downloaded from the workstation 102 to receive the real-time MR data and provide buffer storage, such that no data is lost by data overrun. In some scans, the data acquisition server 112 does little more than pass the acquired MR data to the data processor server 114. However, in scans that require information derived from acquired MR data to control the further performance of the scan, the data acquisition server 112 is programmed to produce such information and convey it to the pulse sequence server 110. For example, during prescans, MR data is acquired and used to calibrate the pulse sequence performed by the pulse sequence server 110.
The data processing server 114 receives MR data from the data acquisition server 112 and processes it in accordance with instructions downloaded from the workstation 102. Images reconstructed by the data processing server 114 are conveyed back to the workstation 102 where they are stored. Real-time images are stored in a data base memory cache (not shown), from which they may be output to operator display 112 or a display 136 that is located near the magnet assembly 124 for use by attending physicians. Batch mode images or selected real time images are stored in a host database on disc storage 138. When such images have been reconstructed and transferred to storage, the data processing server 114 notifies the data store server 116 on the workstation 102. The workstation 102 may be used by an operator to archive the images, produce films, or send the images via a network to other facilities.
The invention is further illustrated in the following Examples which are presented for purposes of illustration and not of limitation.
This example discloses the use of a single amino acid Gd-complex as a modular tool for high relaxivity magnetic resonance (MR) contrast agent development.
Introduction. MRI at high magnetic fields (B0) benefits from an increased signal to noise ratio. For MR probes based on gadolinium (Gd, T1 agents), the inherent relaxivity of tissue also increases with increasing B0. Thus, if probe relaxivity (r1) is field independent, the sensitivity of Gd-based probes should increase with B0. Unfortunately, r1 typically decreases with B0 faster than the increase in baseline T1. However, by controlling the rotational dynamics (τR) of the probe, it is possible to create high relaxivity probes with high r1 at high fields (see
Concept. In order to optimize τR and τM for Gd-based high field imaging, we sought a system with an optimal τM and a tunable τR. Gd(DO3A-monopropionate) metal complexes have a near optimal τM in the range of 10-30 ns. Derivatization of the propionate arm with an alanine provides an easily derivatized, dually anchored metal complex with limited rotational freedom. Using standard Fmoc solid phase peptide synthesis, multimerization and control of τR is attainable (see
Synthesis and Relaxivity (1)—monomeric complex. Fmoc-DOTAla was synthesized in 5 steps and with 15% overall yield. Standard manual solid phase synthesis was employed to afford a monomeric prototype Gd-complex Gd(L1) (see
Synthesis and Relaxivity (2)—multimers. Standard manual solid phase synthesis was employed to afford linear, multimeric structures based on the DOTAla ligand system (Gd(L2), Gd(L3)). On-bead deprotection and cyclization of the cysteine residues provides cyclic systems (Gd(C1), Gd(C2), Gd(C3)) (see
Table 1 summarizes the relaxivity values measured in this example. Main numbers are given per Gd, numbers in parentheses are per molecule values. All numbers were determined at 37° C. using an inversion recovery sequence at concentrations ranging from 0.05 to 0.6 mM/Gd. Table 4 in Example 2 further expands the data of Table 1.
Phantoms at 1.5 T.
Conclusions. We have successfully explored the synthesis and application of the novel Gd chelate DOTAla for mono- and multimeric structures (linear and cyclic). We confirmed the predicted mean water residency time of τM=16.6±1.7 ns, providing us with suitable water exchange kinetics for our purposes. Manual solid phase peptide synthesis allows us to vary size and therefore fine tune τR. The DOTAla system is a versatile single amino acid building block, ideal for the development of high relaxivity T1 agents for high magnetic fields.
This example expands further on and provides additional detail regarding the work disclosed in Example 1.
MR imaging at high magnetic fields benefits from an increased signal to noise ratio, however T1 based MR contrast agents show decreasing relaxivity (r1) at higher fields. High field, high relaxivity contrast agents can be designed by carefully controlling the rotational dynamics of the molecule. To this end, we investigated applications of the alanine analogue of Gd(DOTA), Gd(DOTAla). Fmoc protected DOTAla suitable for solid phase peptide synthesis was synthesized and integrated into polypeptide structures. Gd(III) coordination results in very rigid attachment of the metal chelate to the peptide backbone through both the amino acid sidechain and coordination of the amide carbonyl. Linear and cyclic monomers (GdL1, GdC1), dimers (Gd2L2, Gd2C2) and trimers (Gd3L3, Gd3C3) were prepared and relaxivities were determined at different field strengths ranging from 0.47 T to 11.7 T. Amide carbonyl coordination was indirectly confirmed by determination of the hydration number q for the EuL1 integrated into a peptide backbone, q=0.96±0.09. The water residency time of GdL1 at 37° C. was optimal for relaxivity, τM=17±2 ns. Increased molecular size leads to increased per Gd relaxivity (from r1=7.5 for GdL1 to 12.9 mM−1s−1 for Gd3L3 at 1.4 T, 37° C.). The cyclic, multimeric derivatives exhibited slightly higher relaxivities than the corresponding linearized multimers (Gd2C2: r1=10.5 mM−1s−1 versus Gd2C2-red r1=9 mM−1s−1 at 1.4 T, 37° C.). Overall, all six synthesized Gd complexes had higher relaxivities at low, intermediate and high fields than the clinically used small molecule contrast agent [Gd(HP-DO3A)(H2O)].
Magnetic resonance imaging (MRI) is one of the most important modalities used for non-invasive investigation of disease in the clinic. MRI is the imaging technology of choice whenever high-resolution tissue contrast is required. Another advantage is the use of harmless magnetic fields for MRI as opposed to ionizing radiation in the case of CT.1,2 A large fraction of scans performed in the clinical setting are further enhanced by the use of contrast agents.3 Contrast agents shorten the relaxation times of water molecules in their proximity and increase tissue contrast on relaxation weighted imaging sequences.
Currently, most clinically employed contrast agents are non-target specific, small molecule gadolinium complexes which are able to increase the longitudinal relaxation rate 1/T1 of water protons in the extracellular space.4 The extent to which a contrast agent can enhance relaxation depends on its concentration and its relaxivity (r1), an inherent property of the molecule. Most approved contrast agents have low relaxivities (r1) which makes them effective only at relatively high concentrations (≧0.1 mM).4
There has been a considerable research effort to increase the relaxivity of contrast agents.5, 6-9 Compounds with high relaxivity can be detected at lower doses10, or provide greater contrast at equivalent dose to compounds with lower relaxivity. Additionally, an attachment of a targeting moiety allows for target specific delivery of the contrast.10-12 The clinically approved blood pool agent MS-325 (gadofosveset, Ablavar) is an example of a contrast agent with a high relaxivity13-14, this small molecular compound carries an albumin-targeting moiety and will display an over 8 fold increase in relaxivity at low fields once it is associated with human serum albumin (HSA)15, a blood plasma protein.
While 1.5 T remains the dominant field strength for clinical MRI, there is now a large installed base of 3 T scanners and the major equipment vendors also offer 7 T whole body human scanners. Small animal scanners operate almost exclusively at field strengths of 4.7 T and higher. The primary benefit of high field is the increased signal to noise ratio, which enables greater spatial resolution and reduced acquisition time. In addition, the inherent T1 of tissue increases with increasing magnetic field.16 Thus, a contrast agent with equivalent relaxivity at a high and low field would provide much greater contrast at the high field. However, the relaxivity of many T1-contrast agents decreases more rapidly with applied field than the inherent tissue T1 increases.
Relaxivity above 0.1 T depends on a variety of parameters, some of which are depicted in FIG. 7.19 As the magnetic field increases, the optimal correlation time, τc, for maximum possible relaxivity decreases, as it is inversely dependent on the proton Larmor frequency ωH. While the contribution from the electronic relaxation time (T1e) is negligible at fields above 1.5 T, contributions from the mean water residency time (τM) and the rotational correlation time (τR) become the levers for generating high relaxivity Gd based agents.20
For the design of high field, high relaxivity contrast agents, it is instructive to consider the equation for two site exchange written in terms of inner-sphere water relaxivity, Eq. (3) below, and the Solomon equation, Eq. (4) below, which describes the field dependence of T1 relaxation of the coordinated inner-sphere water hydrogen atoms.3 Equation 3 teaches that the inner-sphere water relaxation time T1M and the water residency time, τM, should be as short as possible. With regards to T1M, equation 4 indicates that the correlation time should be as large as possible, but while still meeting the requirement of ωH τc<1, where ωH is the proton Larmor frequency and C is a constant. For a given Larmor frequency, there is an optimal correlation time. Unless water exchange is exceedingly fast (>109 s−1), the correlation time at 1.5 T and higher will essentially be the rotational correlation time, τR. If τR is very long (nanoseconds and longer), then relaxivity will be very high at low fields, but the condition ωHτc>1 will also occur at lower fields and relaxivity will be low at high fields.
For instance, MS-325 was designed for high relaxivity at low fields (≦1.5 T). Serum albumin binding of MS-325 results in a very long τR resulting in high relaxivity at 1.5 T, but a precipitous decline in relaxivity with increasing field strength.21,22 Small molecule agents with very short correlation times, such as [Gd(DTPA)(H2O)]2− (Magnevist), display a modest relaxivity decrease with increasing field strength but exhibit relatively low relaxivity due to their rapid tumbling.23
We have previously investigated interplay of water exchange and rotational correlation time for Gd-based T1 agents at fields ranging from 0.47 to 9.4 T,21 and showed that the optimal ranges are 5<τM<25 ns and 0.5<τR<2 ns to yield high relaxivity over a range of fields. A number of compounds with a corresponding, intermediate τR value between 0.35-1 ns has been reported;18, 24, 25 however, none of these structures allow for the simple adjustment of τR without sacrificing rigidity of the Gd-complex or complete redesign of the entire scaffold. The work in this example describes synthesis and investigation of a unique, modular system, capable of the construction of a new generation of high relaxivity T1 contrast agents for high magnetic fields. Peptide structure and Gd complex incorporation can be modified using solid phase peptide synthesis, without change of the local complex environment.
Control and optimization of τR requires rigid attachment of the corresponding Gd complex to a molecular construct of appropriate size. Conjugating the Gd complex to a targeting vector or molecular scaffold is typically done through a single linkage, and this results in fast internal motion about that linkage and concomitant lower relaxivity. Tweedle and coworkers introduced the dual anchor strategy,26 which was also employed by Desreux and colleagues to rigidify attachment of the metal complex for the construction of fatty acid derivatized Gd(DOTA)27. A similar, multi-site attachment strategy was employed for the design of metallostars, where a metallic barycenter is used as a point of attachment for multiple Gd(DTTA) type complexes (
The immediate coordination environment around the Gd complex influences important parameters such as kinetic inertness and water exchange kinetics. While q≧2 complexes can provide great relaxivity enhancement due to two or more possible sites of interaction for water molecules with the paramagnetic metal,30-34 only few have the kinetic inertness with respect to Gd dissociation/transchelation required for in vivo applications. For q=1, a myriad of kinetically inert Gd(DOTA) type complexes have been characterized.35, 36
DOTA mono-proponiamide derivatives, where the amide forms a 6-membered chelate ring upon coordination of Gd(III), were found to have a mean water residency time of 10-20 ns (at 37° C.), which is within the ideal range required for our purposes (
For the use of DO3A-N-α-aminopropionate in solid phase peptide synthesis, construction of the corresponding Fmoc-derivative (“Fmoc-DOTAla-tBu3”, compound 6) was required. Fmoc is easily deprotected under mildly basic conditions while the ligand-carboxylates remain protected41; hence, it is more suitable for our purposes than a potential Boc-derivative.5 As cyclen has high inherent basicity, the Fmoc protective group can only be introduced after alkylation of all secondary amines on cyclen.
We employed a synthetic strategy in order to afford compound 6 in 15% over all yield after five synthetic steps (see
As a next step, we aimed to incorporate compound 6 into the linear model sequence H-Cys(Acm)-Gly-DOTAla-Gly-Phe-Cys(Acm)-CONH2 (H3L1, see
Incorporation of a Phe residue was employed to provide a UV handle for detection and purification. Two Cys residues were used as the terminal amino acids, serving as potential sites of secondary structure modification through intramolecular disulfide bond formation. The tert-butyl esters were removed simultaneously with cleavage from the resin using a typical acidic cleavage cocktail (TFA:DDT:TIPS:H2O (9.5:0.25:0.25:0.25)). The crude peptide H3L1 was isolated using ether precipitation. Complexation with either GdCl3.6H2O or EuCl3.6H2O by mixing of aqueous solutions of the metal salt and the crude peptide at pH 3, followed by slow adjustment of the pH to 6.5 using an aqueous 0.1 M NaOH solution yielded the corresponding crude metal complex. The metallopeptide was purified using preparative HPLC.
This class of lanthanide-DOTA derivatives is typically 9-coordinate. If the amide carbonyl from the peptide backbone was coordinated to the lanthanide, then we would expect a single aqua co-ligand, i.e., q=1. The luminescence lifetime of the Eu(III) complex was measured in H2O and D2O. A custom-designed multimodal confocal imaging system built by Yaseen et al.42 was used to measure the luminescence lifetime of the Eu(III) excited state 5D1 as previously reported.43 Luminescence lifetimes were measured and averaged and used for the modified Horrocks equation44 (equation 3), which accounts for the amide donor as one of the ligands.
q
H2O=1.2[(1/τH2O−1/τD2O)−0.325] (3)
The value obtained for hydration number q was 0.96±0.09, which suggests that the carbonyl of the peptide backbone is indeed coordinated to the metal ion.
Water exchange kinetics for the inner-sphere water ligand were determined by measurement of the temperature dependence of the transverse relaxation time T2 of H217O in the presence and absence of GdL1. The data in
At the high field used, τM dominates the scalar correlation time and results in an accurate estimate of water exchange, while the relative contribution of T1e to 17O nuclear relaxation is much lower and this parameter is less well defined, see Table 2. The water residency time (τM=1/kex) was determined to be 17±2 ns at 310 K, which is similar to the Gd-DOTA-monopropionamide derivative reported by Geraldes and coworkers (Table 2, FIG. 12).38 This similar water exchange rate is also consistent with the amide carbonyl as a donor. We further note that this water residency time is in the optimal range for high relaxivity at all field strengths.
310T1e = 160 ± 124 ns and AE‡ = 21 ± 18 kJ mol−1 for GdL1.
310kex × 106 (s−1)
310 TM (ns)
This similar water exchange rate is also consistent with the amide carbonyl as a donor. We further note that this water residency time is in the optimal range for relaxivity at all field strengths.
GdL1 demonstrated that the GdDOTAla moiety could be incorporated into a peptide, and the resultant complex had the expected single inner-sphere water co-ligand with an optimal water exchange rate. However GdL1 is still a rather small molecule with a relatively short τR. In order to increase τR and enhance the molecular relaxivity, we also synthesized dimeric and trimeric structures. The cysteines were either left protected (‘linear’ structures) or were deprotected and used to induce intramolecular cyclization (‘cyclic’ structures) in order to highlight the possibility of secondary structure modification with our approach (see
Multimers H6L2 and H9L3 were furnished using the same synthesis methodology as for the linear, model monomer peptide H3L1. On-bead deprotection of the Acm (acetamidomethyl) protective group on the Cys amino acids using I2 was done in order to afford the cyclicized analogues H6C1, H6C2 and H9C3.46 As cyclization was only 60% complete for compounds H6C2 and H9C3, cyclization was driven to completion using 2% DMSO in H2O at pH 8 (see FIG. 11).47 The Gd complexes GdL1, Gd2L2 Gd3L3, GdC1, Gd2C2 and Gd3C3 were formed and purified using the same methodology as described for the monomer. Isolated yields for the cyclic products were considerably lower due to intermolecular disulfide bond formation resulting in polymeric side products, which are separated by HPLC purification. All Gd complexes were characterized using LC ESI-MS.
The development of new contrast agents requires compounds with high thermodynamic stability and kinetic inertness with respect to Gd dechelation. Tei et al. showed that a GdDOTA-monoproponiamide derivative had a very high stability constant, log KML=20.2,37 and we expected that our system with the same donor set would exhibit similar thermodynamic stability. To address kinetic inertness, we measured the full transchelation of Gd(III) from the complexes GdL1, Gd2L2 Gd3L3 to a DTPA derivative with higher thermodynamic stability. Each of these complexes was challenged with one equivalent of the ligand of MS-325 (MS-325-L) on a per gadolinium basis (e.g. Gd3L3 was challenged with 3 equivalents of MS-325-L).
MS-325-L is a DTPA derivative with a biphenyl moiety that enables easy separation and monitoring of the free ligand from the MS-325 gadolinium complex by HPLC.
Transchelation was monitored using LC-MS, via formation of the MS-325 complex. For comparison, we also measured transchelation from the approved contrast agents [Gd(HP-DO3A)(H2O)] (ProHance, gadoteridol) and [Gd(DTPA)(H2O)]2− (Magnevist, gadopentetate). Although thermodynamically favored, it is apparent from
Per Gd relaxivities were determined by measuring T1 at 37° C. using 20, 60, 200, 400 and 500 MHz spectrometers. Relaxivities for MS-325 (with and without the presence of HSA) as a reference compound with a long τR and [Gd(HP-DO3A)(H2O)] as a reference for very short τR were also measured, and all the relaxivity data is tabulated in Table 4, together with results obtained from literature for the compounds with similar estimated τR. At low fields such as 0.47 and 1.4 T, the compounds with the highest rotational correlation times (MS-325/HSA and the trimers) exhibit the highest relaxivity. Additional rigidity through cyclization seems to provide only minor relaxivity increase for the dimeric and trimeric systems (Gd2C2 and Gd3C3).
At intermediate field (4.7 T), only a moderate decrease in relaxivity is observed for the metallopeptides. In comparison, HSA-associated MS-325 exhibits a peak molecular relaxivity of above 40 mM−1s−1,49 followed by rapid decrease in relaxivity upon increase of the magnetic field. Because we use the rigid GdDOTAla amino acid for multimerization, both the per Gd relaxivity and per molecule relaxivity increase with increased molecular size.
In order to further illustrate this, we imaged a series of phantoms at 4.7 T (
At 9.4 T, the per Gd relaxivities were 6.1 and 6.6 mM−1s−1 for the trimeric metallopeptides. These values are also found to be higher than the relaxivities measured at 9.4 T for previously reported trimeric compounds of similar composition and hydration number.5, 30 For compounds based on q=2 complexes, higher relaxivities can be obtained.17
Investigation of the effect of tertiary structure on relaxivity, was done by examination of the effect of disulfide bond reduction on T1 at 0.47 and 1.41 T. T1 was measured for each sample at 37° C. and then the samples were incubated with 20 eq. TCEP for 30 minutes at room temperature to reduce the intramolecular disulfide bond and give the linear peptide. Subsequently, the T1 values were remeasured and concentrations re-determined in order to calculate relaxivities. A slight decrease in relaxivity (7-14%) was observed for Gd2C2-red (9 mM−1s−1) and Gd3C3-red (11.5 mM−1s−1). Over all, reduction of the disulfide bond has only a slight effect on relaxivity (
We hypothesize that the large Gd-chelate side chain and the Gd(III) coordination by the amide carbonyl imposes defined structure to the peptide that dominates the over-all molecule structures for both the linear and the cyclic multimers. Introduction of a secondary structure modification such as the cyclization has only a marginal influence on the relaxivity. Nevertheless, facile introduction of the disulfide bridge by use of standard peptide synthesis methodology demonstrates the modularity of our system.
The high field relaxivities that we have obtained are consistent with an intermediate rotational correlation time. By assuming that the contributions of second-sphere and outer-sphere water can be estimated from a related q=0 complex,50 we estimate τR of these metallopeptides to be in 150-600 ps range, based on the magnitude and field dependence of their relaxivities. More precise estimates of τR could be obtained by additional relaxation measurements using high resolution NMR with other Ln surrogates of Gd.51 Compared to the other multimers reported in Table 4, our relaxivities are similar. For a specific field strength, the rotational dynamics will dictate the optimal relaxivity. The modular amino acid approach presented here offers the possibility to tune such a high field relaxivity by systematically controlling the size and nuclearity of the complex.
In conclusion, we were able to synthesize a single amino acid Gd chelate, Gd(DOTAla), suitable for solid phase peptide synthesis. The chelate is unique as it provides rigid and stable attachment of the metal complex to the rest of the molecule by using the amido-carbonyl of the corresponding peptide backbone as a point of attachment. Gd(DOTAla) when incorporated into a peptide exhibits one inner-sphere water ligand that has an optimal rate of water exchange for relaxometric purposes. The macrocyclic structure of the chelate provides high thermodynamic stability and kinetic inertness with respect to transchelation or Gd dissociation. The rigid incorporation of Gd(DOTAla) into a peptide scaffold allows design of contrast agents with defined rotational dynamics. Here, we described six new compounds containing 1-3 Gd(DOTAla) per peptide in a linear or cyclic peptide framework. By careful control of the rotational dynamics, it is possible to design contrast agents with high relaxivities at both low and high magnetic fields. These new contrast agents were superior to commercial contrast agents [Gd(HP-DO3A)(H2O)] and MS-325/HSA at high fields. The modularity of design, the ease of solid phase synthesis, high kinetic inertness, and optimal water exchange rate renders the Gd(DOTAla) scaffold a suitable platform for the development of high field T1 agents based on Gd.
1H and 13C NMR spectra were recorded on a Varian 11.7 T NMR system equipped with a 5 mm broadband probe. Purification via HPLC of intermediates toward Fmoc-DOTAla was performed using method A: Injection of crude mixture onto preparative HPLC on a Rainin, Dynamax (column: 250 mm Polaris C18) by using A: 0.1% TFA in water, B: 0.1% TFA in MeCN, flow-rate 15 mL/min, from 5% B to 95% B over 20 minutes. Purification of Gd complexes was performed using method B: Injection of crude mixture onto analytical column (Phenomenex Luna, C18(2) 100/2 mm) using A: water, B: MeCN, flow-rate 0.8 mL/min, 15 min gradient from 2% B to 60% B over 15 min. Monitoring of UV absorption was done at 220 nm. HPLC purity analysis (both UV and MS detection) was carried out on an Agilent 1100 system (column: Phenomenex Luna, C18(2) 100/2 mm) with UV detection at 220, 254 and 280 nm by using a method C: A gradient of 95% A (0.1% formic acid in water) to 95% B (0.1% formic acid in MeCN), flow-rate 0.8 mL/min, 1 over 15 minutes. Kinetic inertness measurements were also carried out using the LCMS agilent system, using method D: A gradient of 95% A (ammonium formate, 20 mM, pH 6.8) with 5% (9:1 MeCN/20 mm ammonium formate) to 95% B (9:1 MeCN/20 mM ammonium formate), flow-rate 0.8 mL/min, 1 over 15 minutes.
The synthesis of ligands was carried out as shown in Schemes 1 and 2. Chemicals were supplied by Aldrich Chemical Co., Inc., and were used without further purification. Solvents (HPLC grade) were purchased from various commercial suppliers and used as received.
Measurements were collected by using the confocal portion of a custom-designed multimodal microscope.42,43 Briefly, a continuous-wave diode laser (l=532 nm, B&W Tek) provided excitation light that was temporally gated by an electro-optical modulator (ConOptics, Danbury, Conn.) with extinction ratio of approximately 200 at 532 nm. The excitation beam passed through several conditioning optics, including a beam expander with pinhole spatial filter, polarizer, shutter, dichroic mirror, scan lens, and tube lens and a 20× magnification objective lens (XLumPlan FL, Olympus, NA=0.95). With the use of a customized control software and galvanometric scanners (Cambridge Technology, Inc. Lexington, Mass., USA), the excitation beam was guided to selected locations in the approximately 600 μm field of view. The emitted luminescence was descanned and collected by using an avalanche photodiode photon counting module (APD, SPCM-AQRH-10, Perkin-Elmer, Waltham, Mass., USA) sampled at 50 MHz with a high-speed DIO card (National Instruments, Austin, Tex., USA). Data were processed by using custom-written software in C and MATLAB (Mathworks, Natick, Mass., USA). Detected luminescent photons were binned into 50 ms long bins, to yield time-dependent phosphorescence decay profiles. With the use of a nonlinear least squares fitting routine, the resulting time-courses were fit with a single-exponential function. A sample's luminescence lifetime is equal to its fitted profile's calculated time constant.
(R)-tri-tert-butyl 2,2′,2″-(10-(3-(benzyloxy)-2-(((benzyloxy)carbonyl)amino)-3-oxopropyl)-1,4,7,10tetraazacyclododecane-1,4,7-triyl)triacetate (4). Cyclen (1.52 g, 8.8 mmol) was dissolved in MeCN (50 mL). K2CO3 (1 eq., 0.61 g) was added and the reaction mixture was preheated to 50° C. (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-((methylsulfonyl)oxy)propanoate (2, 1.8 g, 4.4 mmol) was dissolved in MeCN (20 mL) and added dropwise to the preheated solution. After 16 hours, the precipitate was removed by filtration and the solvent evaporated. The residue was taken up in EtOAc and extracted twice with H2O (80 mL), and once with brine (80 mL). The organic fraction was dried with Na2SO4 and the solvent was evaporated in vacuo to afford the crude mono-cyclen derivative (1.48 g, 3 mmol), which was resuspended in dry MeCN (50 mL) together with K2CO3 (10 eq., 4.24 g). tert-butyl bromoacetate (3.2 eq., 1.45 mL, 1.91 g) was added dropwise and the mixture was stirred for 16 hours at room temperature. The solvent was then removed and the residue was resuspended in EtOAc and extracted with H2O and brine. The organic fraction was collected, dried with Na2SO4 and the solvent was evaporated in vacuo to afford the crude product which was purified using preparative HPLC, method A. Yield: 1.03 g (1.24 mmol, 28%). 1H NMR (CDCl3, 500 MHz, 298 K): δ=7.31-7.30 (m, Bn-H, 10H), 5.14-5.04 (m, CH2-Bn, 4H), 4.75 (brs, α-CH, 1H), 3.75-3.05 (m, cyclen-H/N—CH2—COOtBu, 24H), 1.47-1.42 (m, CH3, 27H); 13C NMR (CDCl3, 125 MHz, 303 K): δ=167.8, 167.7, 160.9, 160.7, 136.2, 134.8, 128.6, 128.5, 128.2, 127.9, 119.5, 117.2, 114.9, 112.6, 83.3, 68.0, 67.2, 55.0, 54.7, 50.9, 50.15, 27.9; LC/MS (ESI+): C44H67N5O10 m/z: calcd. 826.5 [MH+]; found: 826.4 (MH+).
(R)-2-amino-3-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10 tetraazacyclododecan-1-yl)propanoic acid (5). Compound 4 (5.5 g, 6.7 mmol) was dissolved in EtOH (600 mL). Pd/C (2.9 g, 10% w/w) was added to afford a slurry which was subjected to H2 (35 psi) for 3 hours. The Pd/C was filtered off and the filtrate was reduced in vacuo to afford the product (3.85 g, 6.4 mmol) as a colorless oil which was used without further purification in the subsequent reaction step. 1H NMR (CD3OD, 500 MHz, 298 K): δ=4.18 (brs, α-CH, 1H), 4.85-3.15 (m, cyclen-H/N—CH2—COOtBu, 24H), 1.53-1.50 (m, CH3, 27H); 13C NMR (CD3OD, 125 MHz, 303 K): δ=170.4, 161.4, 161.2, 83.4, 54.3, 50.1, 49.1, 26.9; LC/MS (ESI+): C29H55N5O8 m/z: calcd. 602.4 [MH+]; found: 602.5 (MH+).
(R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)propanoic acid (6). Compound 5 (2.395 g, 3.98 mmol) was dissolved in dioxane (60 mL). Na2CO3 (1.27 g, 11.9 mmol, 3 eq) was dissolved in H2O. The two solutions were mixed and cooled to 0° C. Fmoc-Cl (1.125 g, 4.3 mmol) was dissolved in dioxane (5 mL) and added to the reaction mixture. The solution was allowed to warm to room temperature and stirred for 4 hours. The solvent was then removed and the solid residue was dissolved in MeCN. The residual solid was filtered off and the filtrate was purified using preparative HPLC, method A. The product fractions were pooled and the solvent was removed in vacuo to afford the clean product as a white solid (1.81 g, 2.2 mmol, 55%). 1H NMR (CDCl3, 500 MHz, 298 K): δ=7.30-7.26 (m, FmocAr—H, 10H), 4.76 (brs, α-CH, 1H), 4.30 (m, CH2-Fmoc, 2H), 4.15 (q, CH-Fmoc, 1H), 3.75-3.05 (m, cyclen-H/N—CH2—COOtBu, 24H), 1.45-1.36 (m, CH3, 27H); 13C NMR (CDCl3, 125 MHz, 303 K): δ=171.1, 169.7, 143.6, 141.2, 127.8, 127.2, 125.2, 119.9, 84.8, 83.3, 67.5, 54.0, 50.7, 48.3, 46.8, 27.8; LC/MS (ESI+): C44H65N5O10 m/z: calcd. 824.5 [MH+]; found: 824.4 (MH+).
Solid-phase peptide synthesis was carried out manually following standard Fmoc protocols using PEGA Rink amide resin. All peptide sequences were derived from one solid support 0.33 mmol scale using single step couplings of four equivalents of Fmoc-amino acids, two equivalents coupling agent (HATU) and 3 equivalents N-methylmorpholine (NMM) in DMF at room temperature (refer to scheme 2). Coupling with commercial amino acids was completed within 12 hours (Step i), while Fmoc-DOTAla was only used in a 1.5 equivalent excess and allowed to react with the free N terminus of the peptide for 48 hours (Step ii). The coupling step was followed by rinsing with DMF and deprotection with 20% piperidine in DMF for 2 hours. After subsequent thorough rinsing with DMF and dichloromethane, a small aliquot of solid support was removed from the batch and deprotected using cleavage cocktail (TFA:DDT:TIPS:Water (9.5:0.25:0.25:0.25)) room temperature for 2 hours. The resin was filtered off and the filtrate concentrated with a gentle nitrogen flow. The intermediate was precipitated with cold diethyl ether, collected and characterized by ESI-MS. If coupling was found to be complete, the next coupling step was initiated on the main peptide batch. Once a sequence was complete, the corresponding aliquot was removed from the main resin batch and completed by addition of the terminal Fmoc-Cysteine-S-Acm.
For cyclic sequences, treating the resin-bound peptide with 10 equivalents of I2 in DMF for 6 hours completed side chain deprotection with simultaneous cyclicization of the Cys residues (Step iii). After thorough rinsing of the resin with DMF and dichloromethane following the final processing step on-bead (Fmoc deprotection for linear systems, I2 cyclicization for cyclic sequences), the crude peptide was afforded by cleaving from the resin using the acidic cleavage cocktail (see above) and isolated by cold ether precipitation, redissolution in water and lyophilization (Step iv). Because the on-bead cyclization proceeds to only approximately 60% completion, the crude peptide is further cyclicized using 2% DMSO in basic H2O (pH˜7.5). As epimerization occurs on the stereocenter of DOTAla, multiple peaks are detected for the corresponding diastereomers.
H2N—C(Acm)PG-DOTAla-GC(Acm)CONH2 (H3L1), HPLC: R=2.4/3.1 min, MS-ESI: m/z: 1043.4 (calcd. 1043.3) [M+H]+.
H2N—C(Acm)PG-DOTAla-G-DOTAla-GC(Acm)CONH2 (H6L2), HPLC: Rt=1.3/1.48 min, MS-ESI: m/z: 1514.6 (calcd. 1514.5) [M+H]+.
H2N—C(Acm)PG-DOTAla-G-DOTAla-G-DOTAla-GC(Acm)CONH2 (H9L3), HPLC: Rt=1.21/1.35 min , MS-ESI: m/z: 994.0 (calcd. 994.2) [M+2H]2+.
H2N—C(Scycl)PG-DOTAla-GC(Scycl)CONH2 (H3C1), HPLC: R=1.35/1.6 min, MS-ESI: m/z: 898.4 (calcd. 898.3) [M+H]+.
H2N—C(Scycl)PG-DOTAla-G-DOTAla-GC(Scycl)CONH2 (H6C2), HPLC: R=1.1/1.2 min, MS-ESI: m/z: 1372.5 (calcd. 1372.3) [M+H]+.
H2N—C(Scycl)PG-DOTAla-G-DOTAla-G-DOTAla-GC(Scycl)CONH2 (H9C3), HPLC: R=1.24 min, MS-ESI: m/z: 922.95 (calcd. 922.8) [M+2H]2+.
Gadolinium Complex Formation. Complexes were prepared by adding GdCl3 6H2O stock solution to a solution of ligand at pH 3 while stirring. The pH was gradually adjusted to pH 6.5 using 0.1 M NaOH solution. Complete complex formation was checked by LCMS (no residual ligand detectable). The solution was filtered and purified using preparative HPLC, method B. The Eu(III) complex is formed in analogous fashion.
H2N—C(Acm)PG-DOTAla(Gd)-GC(Acm)CONH2 (GdL1), HPLC: Rt=2.9/3.3 min, MS-ESI: m/z: 1197.3 (calcd. 1197.2) [M+H]+.
H2N—C(Acm)PG-DOTAla(Gd)-G-DOTAla(Gd)-GC(Acm)CONH2 (Gd2L2), HPLC: Rt=2.6/3.0 min, MS-ESI: m/z 912.5 (calcd. 912.5) [M+2H]2+.
H2N—C(Acm)PG-DOTAla(Gd)-G-DOTAla(Gd)-G-DOTAla(Gd)-GC(Acm)CONH2 (Gd3L3), HPLC: Rt=3.2 min, MS-ESI: m/z: 1225.95 (calcd. 1225.8) [M+2H]2+.
H2N—C(Scycl)PG-DOTAla(Gd)-GC(Scycl)CONH2 (GdC1), HPLC: Rt=1.13 min, MS-ESI: m/z: 1053.2 (calcd. 1053.4) [M+H]+.
H2N—C(Scycl)PG-DOTAla(Gd)-G-DOTAla(Gd)-GC(Scycl)CON H2 (Gd2C2), HPLC: R=1.25 min, MS-ESI: m/z 840.7 (calcd. 841.5) [M+2H]2+.
H2N—C(Scycl)PG-DOTAla(Gd)-G-DOTAla(Gd)-G-DOTAla(Gd)-GC(Scycl)CONH2 (Gd3C3), HPLC: R=1.35/1.9 min, MS-ESI: m/z: 1153.8 (calcd. 1154.6) [M+2H]2+.
Reduction of disulfide bond for relaxivity measurements. Complex solutions of purified, cyclic Gd complexes (concentrations of 0.1-0.025 mM, 110 μL) in HEPES buffer (50 mM, pH 7.4) were mixed with TCEP solution (20 mM in HEPES, 10 μL) and incubated room temperature. Reduction was checked by LCMS analysis and found to be complete after 30 minutes.
H2N—C(SH)PG-DOTAla(Gd)-GC(SH)CONH2 (GdC1-red), HPLC: Rt=2.35 min, MS-ESI: m/z: 1055.2 (calcd. 1055.2) [M+H]+.
H2N—C(SH)PG-DOTAla(Gd)-G-DOTAla(Gd)-GC(SH)CONH2 (Gd2C2-red), HPLC: R=2.8-3.1 min, MS-ESI: m/z 841.7 (calcd. 842.0) [M+2H]2+.
H2N—C(SH)PG-DOTAla(Gd)-G-DOTAla(Gd)-G-DOTAla(Gd)-GC(SH)CONH2 (Gd3C3-red), HPLC: Rt=3.1-3.3 min, MS-ESI: m/z: 1155 (calcd. 1155) [M+2H]2+.
Measurement of kinetic inertness. Stock solutions of MS-325-L and GdL1, Gd2L2 and Gd3L3 were prepared in 50 mM citrate buffer at pH 3.0. MS-325-L was added to solutions of the Gd complexes and incubated at 37° C. The final concentrations of the metal complexes were 0.1 mM, while the concentration of MS-325-L was adjusted according to the amounts of Gd complexes per metallopeptide present. A 10 μL aliquot was removed for HPLC analysis and analyzed while the remainder of the solution was incubated at 37° C. A 10 μL aliquot was removed and analyzed at 5, 10, 25, 46, 78, 96, 122, 141 and 244 hours. As a reference, Gd(HP-DO3A) was subjected to MS-325-L under same conditions and measured at time points 0.3, 1.5, 4, 6, 8, 24, 168 and 336 hours.
Measurement of relaxivity. Longitudinal relaxation times T1, were measured on Bruker Minispecs mq20 (0.47 T) and mq60 (1.41 T), a Bruker Bioscan horizontal bore 4.7 T, 9.4 T and 11.7 T Varian NMR spectrometers. T1 was measured by using an inversion recovery method with 10 inversion time values ranging from 0.05×T1 to 5×T1. Relaxivity was calculated from a linear plot of 3 or 4 different concentrations (ranging from 0.01 to 0.5 mM, depending on amount of compound isolated) versus the corresponding inverse relaxation times. All samples were measured at 37° C. using either the internal temperature control of the instrument (0.47, 1.41, 9.4 and 11.7 T) or a warm air blower (4.7 T). MS-325/HSA was prepared in a 4.5% w/v solution of HSA (0.66 mM) in PBS. The MS-325 concentration (in presence of HSA) ranged from 0.05 to 0.15 mM.
17O NMR of GdL1 solution for determination of TM. 17O NMR measurements of solutions were performed at 11.7 T on 150 μL samples contained in 2-mm-shigemi tubes inside a 5 mm standard NMR tube on a Varian spectrometer. Temperature was regulated by air flow controlled by a Varian VT unit. 17O transverse relaxation times of distilled water (pH 3) containing 5% enriched 17OH2 or a 6.88 mM solution of GdLl (pH 7.4, 50 mM HEPES buffer) were measured using a CPMG sequence. The concentration of the sample was determined by ICP-MS. Reduced relaxation rates, 1/T2r were calculated from the difference of 1/T2 between the GdL1 sample and the water blank, and then divided by the mole fraction of coordinated water. The temperature dependence of 1/T2r was fit to a 4-parameter model as previously described.22 The Gd—O hyperfine coupling constant, AM, was assumed to be 3.8×106 rad/s,45 the Gd—O distance was assumed to be 3.1 Å.52
This example discloses experimental data for DOTAla modular human serum albumin (HSA) binders.
1H and 13C NMR spectra were recorded on a Varian 500 NMR system equipped with a 5 mm broadband probe. Longitudinal relaxation times, T1, were measured by using the inversion recovery method on Bruker Minispecs. mq20 (20 MHz) and mq60 (60 MHz). Purification via HPLC of intermediates toward Fmoc-DOTAla was performed using method A: Injection of crude mixture onto preparative HPLC on a Rainin, Dynamax (column: 250 mm Kromasil C18) by using A: 0.1% TFA in water, B: 0.1% TFA in MeCN, flow-rate 15 mL/min, 1 over 23 minutes. HPLC purity analysis (both UV and MS detection) was carried out on an Agilent 1100 system (column: Phenomenex Luna, C18(2) 100/2 mm) with UV detection at 220, 254 and 280 nm by using a method B: A gradient of A(0.1% formic acid in water) to 95% B(0.1% formic acid in MeCN), flow-rate 0.8 mL/min, 1 over 15 minutes.
In order to measure HSA binding of the complexes, a 0.1 mM solution (determined by ICP-MS) of the corresponding Gd complex in 4.5% w/v HSA was prepared and pipetted into a Ultrafree-MC Microcentrifuge Filter (NMWL 5,000 Da, PLCC, Millipore). The mixture is incubated at 37° C. for 10 minutes and subsequently centrifuged at 12,000 rpm for 15 minutes. Binding is determined by measurement of Gd content in the filtrate by ICP-MS.
The synthesis of ligands was carried out as shown in Schemes 1, 2 and 3 (see
The free carboxylate (0.05 mmol) was dissolved in DMF (5 mL). HATU (1.5 eq.), HOAt (1.5 eq.) and NMM (2.5 eq) were added and the mixture was agitated for 5 minutes in order to activate the carboxylate. Subsequently, the corresponding free amine (1.5 eq.) was added to the mixture and the solution was stirred at room temperature over night. Reaction control via LCMS (method B, see above) was used in order to evaluate if product was present. The reaction mixture was purified using preparative HPLC (method A). Fractions containing the pure product were pooled, lyophilized and used for the subsequent reaction step after analysis.
The Fmoc-protected intermediate synthon (0.01-0.05 mmol) was dissolved in DMF (1 mL) and added to a solution of solid-support-piperazine (SSP-pip, 10 eq.) suspended in DMF (5 mL). The mixture was stirred at room temperature over night. Reaction control via LCMS (method B, see above) was used in order to evaluate any residual starting material was present. Another batch of SSP-pip (10 eq.) was added if significant amounts of starting material were found to be present. Once the reaction was complete, the reaction mixture was filtered in order to remove the SSP-pip. The filtrate was used without further purification for the next reaction step if procedure 1 followed, or purified using preparative HPLC (method B), if no additional peptide couplings followed. For compounds 3, 6, 9, the solvent is removed and the intermediate used for the deprotection step without further purification. The only contaminant observed (1-((9H-fluoren-9-yl)methyl)piperazine) is removed via filtration of the subsequent reaction mixture.
The starting material (0.01-0.02 mmol) was dissolved in a mixture of dichloromethane and trifluoroacetic acid (1:1, 2 mL) and stirred over night. LCMS provided reaction control. The solvent is removed in vacuo and the product is isolated as the trifluoroacetate salt.
The ligand (0.01 mmol) was redissolved in H2O. The pH was adjusted to 3 and GdCl3.6H2O (0.07 mmol) was added and the pH was adjusted to 6.5 using NaOH (0.1 M). Complexation was found to be complete once less than 5% free ligand was detected, as determined by LCMS (method A).
Tri-tert-butyl 2,2′,2″-(10-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-((2-methoxy-2-oxoethyl)amino)-3-oxopropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (2). 1H-NMR (CDCl3, 500 MHz, ppm): 8.75(s (br), NH), 7.75 (d, 2H), 7.61 (m, 2H), 7.41-7.28 (m, 4H), 4.77 (s(br), 1H), 4.41-3.05 (m, 31H), 1.43-1.39 (m, 27H). 13C-NMR (CDCl3, 125 MHz, ppm): 169.9, 169.3, 161.3, 161.0, 143.7, 143.5, 141.3, 141.2, 127.8, 127.1, 125.2, 119.9, 117.4, 115.0, 113.1, 82.6, 67.5, 54.8, 52.2, 50.9, 46.9, 41.1, 29.7, 28.0. LC-ESI-MS: calcd. for C47H71N6O11: 895.5. Found: 895.5 [M+H]+, Rt=6.36 min.
Tri-tert-butyl 2,2′,2″-(10-(2-amino-3-((2-methoxy-2-oxoethyl)amino)-3-oxopropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (3). 1H-NMR (D2O, 500 MHz, ppm): 3.95 (d, 2H), 3.63 (s, 3H), 3.48-2.2 (m, 24H), 1.42-1.34 (m, 27H). LC-ESI-MS: calcd. for C32H61N6O9: 673.5 Found: 673.5 [M+H]+, Rt=5.02 min.
2,2′,2″-(10-(2-amino-3-((2-methoxy-2-oxoethyl)amino)-3-oxopropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (4). 1H-NMR (D2O, 500 MHz, ppm): 3.64-3.63 (m, 5H), 3.51 (s, 3H), 3.51-2.58 (m, 22H). 13C-NMR (D2O, 125 MHz, ppm): 175.8, 171.6, 170.4, 170.3, 58.1, 56.7, 53.9, 52.9, 52.4, 50.4, 48.9, 46.0, 41.22. LC-ESI-MS: calcd. for C20H37N6O9: 505.25 Found: 505.3 [M+H]+, R=6.1 min (method B, ultra-aqueous column).
Tri-tert-butyl 2,2′,2″-(10-(2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-hydroxy-3,5-diiodophenyl)propanamido)-3-(((S)-1-methoxy-1-oxopropan-2-yl)amino)-3-oxopropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (5). 1H-NMR (CDCl3, 500 MHz, ppm): 7.73 (d, 2H), 7.61-7.54 (m, 4H), 7.41-7.28 (m, 4H), 4.95 (s(br), 1H), 4.39-2.94 (m, 31H), 1.47-1.41 (m, 27H). 13C-NMR (CDCl3, 125 MHz, ppm): 169.9, 161.5, 161.2, 156.6, 152.7, 143.7, 141.2, 141.1, 140.0, 139.9, 127.7, 127.2, 125.4, 125.3, 119.8, 117.4, 115.0, 82.8, 82.4, 67.5, 58.4, 56.9, 55.1, 52.2, 49.3, 48.6, 46.9 41.3, 28.0, 27.9. LC-ESI-MS: calcd. for C56H78I2N7O13: 1310.4. Found: 1310.3 [M+H]+, R=7.2 min.
Tri-tert-butyl 2,2′,2″-(10-(2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-hydroxy-3,5-diiodophenyl)propanamido)-3-(((S)-1-methoxy-1-oxopropan-2-yl)amino)-3-oxopropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (6). 1H-NMR (CDCl3, 500 MHz, ppm): 7.59 (s, 2H), 7.41-7.28 (m, 4H), 4.95 (m, 2H), 3.9-2.94 (m, 32H), 1.50-1.44 (m, 27H). LC-ESI-MS: calcd. for C41H68I2N7O11. 1088.3 Found: 1088.3 [M+H]+, Rt=5.2 min.
2,2′,2″-(10-(2-(2-amino-3-(4-hydroxy-3,5-diiodophenyl)propanamido)-3-((2-methoxy-2-oxoethyl)amino)-3-oxopropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (7). 1H-NMR (D2O, 500 MHz, ppm): 7.69 (s, 2H), 4.62 (d, 1H), 4.18 (s, 1H), 3.94-2.80 (m, 30H). 13C-NMR (D2O, 125 MHz, ppm): 171.0, 170.6, 170.1, 160.8, 160.5, 154.9, 140.8, 140.3, 129.4, 117.5, 115.1, 84.4, 84.0, 61.9, 56.1, 53.8, 53.1, 51.5, 51.3, 40.9. LC-ESI-MS: calcd. for C29H44I2N7O11: 920.1 Found: 920.1 [M+H]+, Rt=3.9 min.
Tri-tert-butyl 2,2′,2″-(10-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-oxo-3-(((R)-1-phenylethyl)amino)propyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (8). 1H-NMR (CDCl3, 500 MHz, ppm): 8.33 (s, 1H), 8.25 (s, 1H), 7.74 (m, 2H), 7.63-7.59 (m, 2H), 7.41-7.16 (m, 9H), 5.01 (m, 1H), 4.69(s, 1 H), 4.39-2.21 (m, 3H), 3.72-2.92 (m, 25H) 1.47-1.42 (m, 31H). 13C-NMR (CDCl3, 125 MHz, ppm): 169.4, 161.3, 161.0, 143.5, 141.3, 128.6, 128.5, 127.7, 127.2, 127.1, 126.2, 126.1, 125.2, 119.9, 117.4, 82.6, 55.1, 53.4, 51.0, 49.9, 49.8, 46.9, 28.9, 22.0. LC-ESI-MS: calcd. for C52H75N6O9: 927.5 Found: 927.4 [M+H]+, Rt=6.76 min.
Tri-tert-butyl 2,2′,2″-(10-(2-amino-3-oxo-3-(((R)-1-phenylethyl)amino)propyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (9). 1H-NMR (D2O, 500 MHz, ppm): 7.26-7.21 (m, 5H), 4.62 (d, 1H), 4.81 (s, 1H), 3.41-2.80 (m, 26H), 1.35-1.25 (m,31H LC-ESI-MS: calcd. for C37H65N6O7: 705.5 Found: 705.4 [M+H]+, Rt=5.5/5.7 min.
2,2′,2″-(10-(2-amino-3-oxo-3-(((R)-1-phenylethyl)amino)propyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (10). 1H-NMR (D2O, 500 MHz, ppm): 7.45-7.37 (m, 5H), 4.98-4.96 (m, 1H), 3.51 (m, 1H), 3.19-2.26 (m, 23H), 1.51 (t, 3H). 13C-NMR (D2O, 125 MHz, ppm): 179.0, 176.6, 128.8, 128.7, 126.0, 125.8, 59.3, 49.4, 21.1. LC-ESI-MS: calcd. for C25H41N6O7: 537.3 Found: 537.3 [M+H]+, Rt=1.06/ 1.21 min.
Tri-tert-butyl 2,2′,2″-(10-(2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-hydroxy-3,5-diiodophenyl)propanamido)-3-oxo-3-(((R)-1-phenylethyl)amino) propyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (11). 1H-NMR (CDCl3, 500 MHz, ppm): 7.71-7.18 (m, 15H), 4.41-3.95 (s(br), 1H), 4.81 (m, 1 h) 4.39-2.74 (m, 29H), 1.51-1.25 (m, 31H). 13C-NMR (CDCl3, 125 MHz, ppm): 172.0, 171.0, 169.9, 161.4, 155.8, 152.9, 143.8, 141.1, 140.0, 139.8, 128.5, 127.7, 127.6, 127.2, 125.9, 125.2, 125.1, 120.0, 119.9, 119.8, 83.2, 82.0, 67.3, 56.9-46.9 (9 broad peaks), 28.0, 22.3. LC-ESI-MS: calcd. for C61H82I2N7O11: 1342.4. Found: 1342.3 [M+H]+, Rt=7.44 min.
Tri-tert-butyl2,2′,2″-(10-(2-(2-amino-3-(4-hydroxy-3,5-diiodophenyl)propanamido)-3-oxo-3-(((R)-1-phenylethyl)amino)propyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (12). 1H-NMR (CD3OD, 500 MHz, ppm): 7.64 (d, 2H), 7.27-7.15 (m, 5H), 5.02 (d, 1 H), 4.10-2.85 (m, 28H), 1.51-1.25 (m, 30H). LC-ESI-MS: calcd. for C46H72I2N7O9: 1120.3 Found: 1120.2 [M+H]+, Rt=5.6/5.8 min.
2,2′,2″-(10-(2-(2-amino-3-(4-hydroxy-3,5-diiodophenyl)propanamido)-3-oxo-3-(((R)-1-phenylethyl)amino)propyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (13). 1H-NMR (CD3OD, 500 MHz, ppm): 8.38 (d, 1H) 7.65 (d, 2H), 7.33-7.24 (m, 5H), 4.98 (d, 1H), 4.76-4.03 (m, 5H), 3.64-2.50 (m, 18H), 1.51-1.39 (t, 3H). 1H-NMR (CD3OD, 125 MHz, ppm): 169.7, 169.2, 160.2, 159.9, 154.9, 142.9, 140.6, 129.7, 128.1, 126.8, 125.9, 117.4, 114.8, 89.4, 81.2, 81.1, 53.8, 53.3, 51.3, 50.9, 49.0, 48.2, 34.3. LC-ESI-MS: calcd. for C34H48I2N7O9: 952.2 Found: 952.2 [M+H]+, Rt=4.8/ 4.9 min.
2,2′,2″-(10-(2-acetamido-3-oxo-3-(((R)-1-phenylethyl)amino)propyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (14). tri-tert-butyl 2,2′,2″-(10-(2-amino-3-oxo-3-(((R)-1-phenylethyl)amino)propyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (9, 11 mg, 0.015 mmol), was dissolved in dichloromethane (2 mL). Triethylamine (2 eq., 5 μL), followed by acetic anhydride (5 eq.) was added and the reaction was stirred over night at room temperature. LCMS analysis indicated complete conversion of starting material. The product was purified using method A. The fractions containing product were pooled and lyophilized to afford the protected precursor (1 mg, 0.0015 mmol, 10% yield after purification). Subsequent deprotection of the tert-butyl acetate moieties yielded the final product 14. 1H-NMR (CD3OD, 500 MHz, ppm): 8.4 (s, 1H) 7.29-7.15 (m, 5H), 4.95 (s, 1 H), 4.01-2.85 (m, 24H), 1.91 (s, 3H), 1.39 (t, 3H). 1H-NMR (CD3OD, 125 MHz, ppm): 178.3, 154.4, 147.3, 143.2, 128.2, 126.8, 125.9, 56.0, 55.7, 53.3, 50.6, 49.1, 48.4, 29.3, 15.6. LC-ESI-MS: calcd. for C27H43N6O8: 579.3 Found: 579.3 [M+H]+, Rt=1.7 min.
(S)-tert-butyl 2-amino-3-(4-hydroxy-3,5-diiodophenyl)propanoate (15). (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(4-hydroxy-3,5diiodophenyl)propanoic acid (0.67g, 1 mmol) was dissolved in tert-butyl acetate (7 mL). Perchloric acid (1.5 eq, 90 μL) was added and the solution was stirred at room temperature over night. The acid was neutralized with a saturated aqueous solution of NaHCO3 and the product was extracted three times with dichloromethane. The organic layer was collected, dried with Na2SO4 and concentrated. The crude product was subsequently purified with a 0-20% gradient of EtOAc in Hexanes on a silica combiflash column. The intermediate product is isolated as yellow oil, which solidifies upon standing. This intermediate was taken up in DMF and the Fmoc protective group was removed according to general procedure 2. The deprotected synthon (15) was separated by filtration of the solid support beads and added to the subsequent reaction without further purification steps.
Tri-tert-butyl 2,2′,2″-(10-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(((S)-1-(tert-butoxy)-3-(4-hydroxy-3,5-diiodophenyl)-1-oxopropan-2-yl)amino)-3-oxopropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (16). 1H-NMR (CDCl3, 500 MHz, ppm): 7.71-7.26 (m, 10H), 4.47-2.90 (m, 29H), 1.48-1.25 (m, 27H). 13C-NMR (CDCl3, 125 MHz, ppm): 169.9, 160.7, 160.5, 152.6, 152.6, 143.4, 141.3, 141.2, 139.9, 129.7, 128.3, 127.8, 127.1, 127.1, 125.2, 124.9, 120.1, 116.5, 114.2, 83.2, 82.0, 67.5, 67.3, 55.1, 54.5, 54.1, 46.9, 46.9, 46.7, 27.8. LC-ESI-MS: calcd. for C57H81I2N6O12: 1295.4. Found: 1295.4 [M+H]+, Rt=7.9 min.
Tri-tert-butyl 2,2′,2″-(10-(3-(((S)-1-(tert-butoxy)-3-(4-hydroxy-3,5-diiodophenyl)-1-oxopropan-2-yl)amino)-3-oxo-2-((R)-2-phenylpropanamido)propyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (18). 1H-NMR (CDCl3, 500 MHz, ppm): 8.07 (d, 1H), 7.53-7.26 (m, 7H), 4.86 (m, 1H), 4.78 (m, 1H) 4.47-2.90 (m, 26H), 1.48-1.25 (m, 30H). LC-ESI-MS: calcd. for C51H79I2N6O11: 1205.4. Found: 1205.3 [M+H]+, Rt=7.3 min.
2,2′,2″-(10-(3-(((S)-1-carboxy-2-(4-hydroxy-3,5-diiodophenyl)ethyl)amino)-3-oxo-2-((R)-2-phenylpropanamido)propyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (19). 1H-NMR (CDOD3, 500 MHz, ppm): 7.97 (d, 1H), 7.61-7.28 (m, 7H), 4.66 (m, 1H), 4.02-2.61 (m, 26H), 1.59 (m, 3H). LC-ESI-MS: calcd. for C35H47I2N6O11: 981.1. Found: 981.1 [M+H]+, Rt=5.4 min.
2,2′,2″-(10-(3-(((1-Carboxy-2-(4-hydroxy-3,5-diiodophenyl)ethyl)-amino)-2-(2-(4-isobutylphenyl)propanamido)-3-oxopropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic Acid, (8). 1H NMR (D2O, 500 MHz, ppm): 7.52 (d, 2H), 7.16-7.00 (m, 4H), 4.48 (m, 1H), 3.86-2.85 (m, 22H), 1.78 (m, 2H), 1.78 (m, 1H), 1.21 (m, 3H) 0.81 (m, 6H). 13C NMR (D2O, 125 MHz, ppm): 171.0, 170.6, 161.2, 153.2, 142.1, 140.6, 139.9, 129.3, 128.8, 128.1, 126.8, 83.9, 65.1, 53.4, 34.6, 30.0, 29.4, 27.4, 24.7, 22.3, 21.5, 21.3. LC-ESI-MS calcd for C39H55I2N6O11: 1037.2. Found: 1037.1 [M +H]+. Looking at
2,2′,2″-(10-(2-Carboxy-2-(2-(4-isobutylphenyl)propanamido)-ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic Acid, (9). 1H NMR (D2O, 500 MHz, ppm): 7.26 (d, 2H), 7.20 (d,2H), 3.73-2.83 (m, 26H), 2.43 (m, 2H), 1.78 (t, 1H), 1.41 (d, 3H),0.81 (m, 6H). 13C NMR (D2O, 125 MHz, ppm): 177.5, 176.9, 141.9, 138.5, 130.1, 127.3, 70.2, 54.4, 46.5, 44.8, 30.7, 27.8, 22.8. LC-ESI-MS calcd for C30H48N5O9: 622.3. Found: 622.4 [M +H]+. Looking at
This example discloses experimental data for 64CuDOTAla.
GdL1 was dissolved in a pH 3 citrate (50 mM) to afford a 0.1 mM solution and incubated for 14 days at 37° C. Within this time, slow transchelation of GdL1 to Gd(citrate) took place. This solution was then incubated with 64CuCl2 (0.2 mCi) at room temperature and the pH was increased to 9 using 0.1 M NaOH solution and the solution was analysed using HPLC (Method C: Phenomenex C18 column, 150×4.6 mm, 5 mycron; Solvent A: H2O with 0.1% TFA, solvent B: Acetonitrile with 0.1% TFA. Gradient: 0-80% B in 12 min, flow: 1 mL/min). Analysis indicated re-complexation of Gd, as well as incorporation of 80% of the free 64Cu into the chelate. Rr 64CuL1 : 4.3 min.
The heptadentate ligand, CyPic3A, was designed to chelate Gd(III) in a stable fashion while allowing for a hydration state of q>1 (
CyPic3A possesses stabilizing structural factors that compensate for reduced denticity. Trans-1,2-diaminocyclohexane was incorporated into the ligand framework. Exchanging 1,2-diaminoethane for this rigidified linkage has been shown to increase the stability and inertness of lanthanide(III) complexes significantly.17,18 The bidentate 2-picolinate functionality is similarly stabilizing.19,20 These two moieties were fused to comprise the ligand backbone.
Coupling of mono-BOC protected trans-1,2-diaminocyclohexane (XIV)21 and methyl 6-formylpyridine-2-carboxylate (XII)22 (prepared in one and two steps, respectively) via reductive amination produced the BOC-protected backbone XV in high yield. Deprotection of XV followed by alkylation of the two amines afforded O-protected CyPic3A (XVII). O-deprotection yielded the ligand, isolated as CyPic3A•2TFA. In total, this straightforward synthesis required seven steps, each affording high yields and few requiring chromatography (see below). Complex formation occurred rapidly in water upon addition of GdCl3.6H2O followed by adjustment of the pH to 6.5. It is important to note that 2,6-pyridinedicarboxylate, from which XII is derived, is a highly modular synthon.23,24 Thus, it is envisioned that bifunctional analogues of CyPic3A can be prepared.
The bis(aqua) hydration state of [Gd(CyPic3A)(H2O)2]− was confirmed by analogy through luminescence lifetime experiments on the Eu(III) congener measured in H2O vs. D2O.25,26 Following excitation at 396 nm, the emission intensity at 616 nm was monitored with respect to time to determine the first order rate constant. There is an empirical relationship that relates the difference in luminescence decay rates measured in H2O and D2O to the hydration number.27 It was determined that q=2.09 using this method.
The mean residency time of the coordinated H2O molecules was also measured by recording the 17O transverse relaxation (T2) times of solvent H2O between 278 and 363 K in the presence and absence of [Gd(CyPic3A)(H2O)2]−. The reduced relaxation rate (1/T2r) is this relaxation rate difference normalized to the mole fraction of water coordinated to the Gd(III). A four-parameter fit to this data yielded water exchange kinetic parameters and an estimate of the electronic relaxation time (T1e).6,28,29 The water residency time at 310 K)(Tm310) was determined to be 14±1 ns, and this very short residency time is optimal for relaxivity applications.
The relaxivity of [Gd(CyPic3A)(H2O)2]− is consistent with two inner-sphere water ligands. At 310 K in pH 7.4 50 mM HEPES buffer, r1=5.70 mM−1 s−1 at 1.41 T (7.53 mM−1s−1 at 298 K). This is 75% higher than [Gd(DTPA)(H2O)]2− measured under these conditions (see below for detailed table of r1 values). To probe the effects of potentially coordinating anions, nwas measured in the presence of 20 equiv. carbonate or L-lactate and also in 25 mM phosphate buffer (pH 7.4). The results are summarized in
As a means of assessing thermodynamic stability relative to other Gd(III)-complexes, [Gd(CyPic3A)(H2O)2]− was challenged with DTPA in pH 7.4 25 mM Tris buffer. CyPic3A possesses a strong chromophore at 280 nm and the relative distributions of complex and free ligand are easily monitored by LC-MS. From this information it was possible to deduce equilibrium constants (Kcomp) for the competition reaction described in Equation 5,31 where L=DTPA and L′=challenging chelator. A Kcomp of 0.17 at was obtained at pH 7.4. This indicates that the affinity of CyPic3A for Gd(III) is about 5-fold lower than DTPA at pH 7.4.
Kinetic inertness was also taken into account in the evaluation of potential probes. In this regard, [Gd(CyPic3A)(H2O)2]− was challenged with 1 equiv. [ZnPO4]− as a slurry in pH 7.0 phosphate buffer at 310 K (see below). Several clinically utilized Gd(III) complexes have been benchmarked relative to one another in this manner.37 As Gd(III) replaced by Zn(II) precipitates from the solution as GdPO4, the relaxation rate 1/T1 decreases. In this method, the time required to reach 80% of the initial 1/T1 value was utilized as an index of kinetic inertness. A time to 80% of the initial 1/T1 of 95 min was measured for [Gd(CyPic3A)(H2O)]−, 124 minutes for [Gd(DTPA-BMA)(H2O)] and 383 minutes for [Gd(DTPA)(H2O)]2−. As with the thermodynamic stability of [Gd(CyPic3A)(H2O)2]− at pH 7.4, the observed kinetic stability also compared well to clinically used agents.
All chemicals and solvents were purchased commercially and used without further purification. NMR spectra were recorded on either a 500 MHz or 400 MHz Varian spectrometer equipped with a 5 mm broadband probe. Chemical shifts are reported in 6 (ppm). For1H NMR spectra, the peak from residual protio solvent was used as an internal reference.1 For 13C NMR spectra, the solvent peaks were used as an internal reference, except when acquired in D2O, in which case dioxane was used as the internal reference.1 Liquid chromatography-electrospray mass spectrometry (LC-MS) was performed using an Agilent 1100 Series apparatus with an LC/MSD trap and Daly conversion diode detector with UV detection at 220, 254 and 280 nm. Characterization of CyPic3A, [Gd(CyPic3A)(H2O)2]−, [Eu(CyPic3A)(H2O)2]− and ligand competition experiments were performed using a Kromasil C18 reversed-phase column (250 mm×4.6 mm) by the following method: the mobile phase was a mixture of 10 mM aqueous ammonium formate (eluent A) and a solution of acetonitrile/ 10% 10 mM aqueous ammonium formate (eluent B). Starting from 5% B, the fraction of B increased to 95% over 12 minutes. The column was washed with 95% B for 2 minutes and than ramped to 5% B. The system was re-equilibrated at 5% B. Reversed-phase semi-preparative purification was performed on the Rainin Dynamax HPLC system with UV detection at 254 nm using a Kromasil C4 (250×21.8 cm) column. The method used for purification is as follows: the mobile phase was a mixture of water (eluent A) and acetonitrile (eluent B), each containing 0.1% TFA. Starting from 5% B, the fraction of B increased to 25% over 23 minutes. The column was washed with 95% B for 2 minutes and then ramped to 5% B. The system was re-equilibrated at 5% B. Gadolinium concentrations were determined using an Agilent 7500a ICP-MS system. All samples were diluted with 0.1% Triton X-100 in 5% nitric acid containing 20 ppb of Lu (as internal standard). The ratio of Gd (157.25)/Lu (174.97) was used to quantify the Gd concentration. A linear calibration curve ranging from 0.1 ppb to 200 ppb was generated daily for the quantification. pH was measured using a ThermoOrion pH meter connected to a VWR Symphony glass electrode. Ultrafiltration was performed using Ultrafree-MC Microcentrifuge filters with a 5,000 Da cut-off PLCC cellulosic membrane. Incubation of samples was performed using a New Brunswick Scientific Inova4000 Incubator Shaker.
Relaxivity measurements were performed on a Bruker mq60 or Bruker mq20 Minispec at 1.41 T and 0.47 T, respectively, and 37° C. Longitudinal (T1) relaxation was acquired via an inversion recovery experiment on 10 inversions of duration ranging between 0.05×T1 and 5×T1. Relaxivity (r1) was determined from the slope of a plot of 1/T1 vs. [Gd] for at least 4 concentrations of Gd(III). The transverse (T2) relaxation times of 17O were acquired at 500 MHz using a CPMG pulse sequence at temperatures ranging from 278 to 368 K. Reduced relaxation rates (1/T2r) were calculated by dividing the [Gd(CyPic3A)(H2O)2]− imparted increase in 1/T2 relative to neat H2O at pH 3 by the mole fraction of coordinated water molecules. This data was plotted against reciprocal temperature (1000/T (K−1)) and fit to a four-parameter model as described previously.2 The Gd—O hyperfine coupling constant, A/h, was assumed to be 3.79×106 rad/s.3 Samples were prepared in neat H2O adjusted and enriched with a small amount of H217O.
Luminescence lifetime measurements were recorded on a Hitachi f-4500 fluorescence spectrophotometer on samples containing ˜50 mM Eu(III). Samples in D2O were lyophilized and reconstituted three times to ensure minimization of residual protio solvent. Excitation was achieved at 396 nm and emission was recorded at 616 nm. A total of 80 replicates were acquired for each sample and the results averaged. Temporal resolution was set to 0.04 ms (0-20 ms) and the PMT voltage was set to 400 V. The luminescent lifetimes were ascertained though monoexponential fits of the data.
Mixtures of precisely defined concentrations of [Gd(CyPic3A)(H2O)2]− and DTPA or MS-325-L were prepared in pH 4 citrate buffer (25 mM) and pH 7.4 Tris buffer (25 mM). Equilibration of each solution was monitored by LC-MS. Relative distributions of [Gd(CyPic3A)(H2O)2]− and CyPic3A were determined by integrating the corresponding absorbance traces at 280 nm. MS-325 and MS-325-L were analyzed analogously at 220 nm. Kcomp values determined between [Gd(DTPA)(H2O)]2− and various ligands were determined from conditional formation constants (Kcond)4 values taken from the literature.6-16 Direct comparisons were only made between Kcond values determined under identical conditions.
Modified conditions of Muller and co-workers were used for this experiment.11,12 Solutions containing 2.5 mM [Gd(CyPic3A)(H2O)2]− and 2.5 mM Zn(OTf)2 were combined in pH 7.0 phosphate buffer (50 mM) and placed in an incubator shaker set to 310 K. Progress of the transchelation was monitored via the decrease in 1/T1 with time. Prior to measurement, aliquots were placed in small glass inserts and centrifuged to bring all insoluble material to the bottom so as not to interfere with the measurement.
The synthesis comprised the following steps:
N—BOC—N′-((6-methylpicol-2-yl)methyl)-trans-1,2-diaminocyclohexane (3). A batch of 349 mg (1.63 mmol) N—BOC-trans-1,2-diaminocyclohexane (1)14 was added to 269 mg (1.63 mmol) of methyl 6-formylpyridine-2-carboxylate (2)15 stirring in 10 mL MeOH. Small aliquots of this reaction were removed and concentrated to dryness for NMR analysis to confirm full Schiff base formation. After 2 hours, the reaction was cooled to 0° C. and 66 mg (1.75 mmoL) NaBH4 was added in 1 mL MeOH forming a deep red solution within minutes. After 2 hours of stirring at 0° C., the reaction was quenched with satd. NaHCO3(aq) and MeOH was removed via rotary evaporation. The volume of the resultant solution was doubled via addition of CH2Cl2 and the reaction was brought to pH 7 using 1M HCl(aq). The organic layer was separated the aqueous phase washed 3× with CH2Cl2. The CH2Cl2 was pooled, dried over Na2SO4 and concentrated to 502 mg (1.38 mmol, 85%) of 3 as a yellow oil. 1H NMR (CDCl3, 400 MHz), δ (ppm): 7.97 (d, 1H), 7.75 (t,1H), 7.54 (d, br, 1H), 5.18 (d, br, 1H, NH), 4.08 (d, 1H), 3.99-3.95 (m, 4H), 3.32 (m, br, 1H), 2.37 (m, 1H), 2.09-2.01 (m, 2H), 1.66-1.62 (m, 2H), 1.41 (s, 9H), 1.29-1.05 (m, 4H). 13C NMR (CDCl3, 100 MHz), δ (ppm): 165.95, 161.82, 156.21, 147.24, 137.33, 125.70, 123.35, 79.07, 64.67, 60.42, 54.75, 51.28, 33.18, 31.97, 28.49, 24.97, 24.61. ESI+ (M +H+) m/z=364.3; calcd: 364.2.
N-((6-methyl picol-2-yl)methyl)-trans-1,2-diaminocyclohexane (4). A batch of 611 mg (1.68 mmol) 3 was dissolved in 3 mL each CH2Cl2: TFA. After 45 minutes, the reaction mixture was concentrated to a pink oil which was taken up in 30 mL CH2Cl2 and stirred over a large excess of K2CO3. After 3 hours, the resultant light yellow solution was filtered and concentrated to 266 mg (1.00 mmol, 60%) of 4 as a yellow oil. It should be noted that the filtrate often contained very little product when the reaction was performed on a larger scale; when this occurred, the slurry containing product and K2CO3 could be carried directly through to the next step (assuming 100% product) and afforded excellent yields. 1H NMR (CDCl3, 500 MHz), δ (ppm): 8.00 (d, 1H), 7.81 (t, 1H), 7.67 (d, 1H), 4.17 (d, 1H), 4.40-3.96 (m, 4H), 2.45 (m, 1H), 2.13 (m, 2H), 1.92-1.67 (m, 7H, NH2 and NH found as broad resonance), 1.30-1.07 (m, 4H). 13C NMR (CDCl3, 125.7 MHz), δ (ppm): 166.08, 161.88, 147.42, 137.47, 125.80, 123.55, 63.95, 55.53, 52.52, 36.14, 31.74, 35.39, 25.33. ESI+ (M+H+): m/z=264.2; calcd: 264.2.
N-((6-methylpicol-2-yl)methyl)-N,N′,N′-tri(tert-butylacetate)-trans-1,2-diaminocyclohexane (5). To 266 mg (1.13 mmol) of 4, 535 mg (3.22 mmol) of potassium iodide and 922 mg (7.13 mmol) diisopropylethylamine stirring in 3 mL DMF was added 732 mg (3.75 mmol) tert-butyl bromoacetate at RT. After 16 hours, the resultant yellow, heterogeneous solution was diluted with 50 mL CH2Cl2, washed with satd. K2CO3(aq), copious water and brine. The organic portion was than dried with Na2SO4 and concentrated to a yellow oil. After chromatography on silica gel using 9:1 to 3:1 hexane: EtOAc, 2.600 g (4.29 mmol, 75%) of 5 was isolated as a yellow oil. 1H NMR (CDCl3, 500 MHz), δ (ppm): 8.05 (d, 1H), 7.92 (t, 1H), 7.76 (t, 1H), 4.12 (d, 1H), 3.94 (s, 3H), 3.80 (d, 1H), 3.45-3.20 (m, 6H), 2.62 (m, br, 1H), 2.53 (t, br, 1H). 2.07-1.99 (m, 2H), 1.66 (s, br, 2H), 1.36 (s, 27H), 1.09-1.00 (m, 4H). 13C NMR (CDCl3, 125.7 MHz), δ (ppm): 171.56, 171.49, 166.05, 161.57, 146.76, 137.37, 127.89, 123.57, 80.76, 80.54, 63.18, 61.70, 58.13, 55.75, 52.83, 28.07 (C(CH3) peaks are coincidental), 26.78, 25.81, 25.66, 18.38. ESI+ (M+H+): m/z=606.4; calcd: 606.4.
N-((6-methylpicol-2-yl)methyl)-N,N′,N′-triacetate-trans-1,2-diaminocyclohexane (CyPic3.2TFA). To a batch of 173 mg (0.29 mmol) 5 in 4 mL 1:1 THF: H2O was added 28 mg (1.17 mmol) lithium hydroxide. After 4 hours stirring at room temperature, the reaction was concentrated to dryness and the resultant residue re-dissolved in 8 mL TFA and stirred at room temperature. After 16 hours, the reaction mixture was concentrated to dryness and purified by preparative HPLC using the method described above (general methods). After pooling of the fractions containing product followed by lyophilization, 84 mg CyPic3A•2TFA was isolated as a white powder. The NMR spectra of the isolated ligand affords broad and ill-resolved peaks when dissolved in D2O untreated. 1H NMR (D2O, 500 MHz), δ (ppm): 8.53 (t, 1H), 8.31 (d, 1H), 8.14 (s, br, 1H), 4.60-3.03 (m, br, 10H), 2.28 (d, 1H), 2.13 (s, br, 1H), 1.85 (s, br, 2H), 1.51-1.26 (m, br, 4H). 1H NMR (D2O, pH>10, 500 MHz), δ (ppm): 7.87 (t, 1H), 7.79 (d, 1H), 7.63 (d, 1H), 3.71 (q, 2H), 3.40 (d, 1H), 3.32 (d, 1H), 2.99 (d, 1H), 2.73 (d, 1H), 2.58-2.49 (m, 2H), 2.39 (t, 1H), 2.15-2.07 (m, 2H), 1.90 (d, 1H), 1.71 (t, 2H), 1.18-1.09 (m, 3H), 0.90 (q, 1H). 13C NMR (D2O, pH≧10, 125.7 MHz), δ (ppm): The 13C resonances were stronger and more visible at alkaline pH: 181.76, 181.44, 181.19, 174.03, 157.62, 153.67, 139.04, 127.42, 122.78, 61.44, 59.00, 58.02, 57.30, 54.23, 52.29, 25.63, 25.50, 24.72, 24.59. ESI+ (M+H+): m/z=424.2; calcd: 424.2
[Gd(CyPic3A)(H2O)2]−. To a batch of 54.5 mg (0.0837 mmol) CyPic3A•2TFA in H2O was added 31.1 mg (0.0837 mmol) of GdCl3.6H2O and the solution adjusted to pH 6.5. Full chelation was affirmed by the testing of small aliquots of this solution in Arsenazo III (0.01 mM in 0.15 M pH 7 ammonium acetate buffer). ESI+ (MW+2H+): m/z=579.0; calcd: 579.1. Less than 1% free ligand was observed by LC-MS.
[Eu(CyPic3A)(H2O)2]−. To a batch of 54.8 mg (0.0841 mmol) CyPic3A•2TFA in H2O was added 24.3 mg (0.0663 mmol) of EuCl3.6H2O was added and the solution adjusted to pH 7.2. Analysis by LC-MS revealed full chelation with a slight excess of ligand species present. This was done to ensure against free Eu(III) during the luminescence measurements. ESI+ (MW+2H+): m/z=574.0; calcd: 574.1
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Example 6 has the following experimental details: (i) DOTAlaP-derivatives: Synthesis of compounds; (ii) DOTAlaP derivatives: Relaxivity; (iii)
DOTAlaP derivatives: Results of Eu-luminescence lifetime derived hydration number; (iv) DOTAlaP derivatives: Results of 17O-NMR derived water exchange; (v) Modified trimer: Synthesis protocol; (vi) Animal model used for trimer/Gadovist imaging; (vii) In vivo imaging protocol: trimer and Gadovist; and (viii) In vivo imaging protocol: ‘Ibuvist’ and ‘P-Ibuvist’.
General materials and methods. General Methods and Materials. 1H and 13C NMR spectra were recorded on a Varian 11.7 T NMR system equipped with a 5 mm broadband probe. Purification via HPLC of intermediates toward DOTAlaP derivatives was performed using method A: Injection of crude mixture onto preparative HPLC on a Rainin, Dynamax (column: 250 mm Phenomenex C18) by using A, 0.1% TFA in water; B, 0.1% TFA in MeCN, flow-rate 15 mL/min, from 5% B to 95% B over 20 minutes. HPLC purity analysis (both UV and MS detection) was carried out on an Agilent 1100 system (column: Phenomenex Luna, C18(2) 100/2 mm) with UV detection at 220, 254, and 280 nm by using method B: Injection of crude mixture onto analytical column (Phenomenex Luna, C18(2) 100/2 mm) using A, water; B, MeCN, flow-rate 0.8 mL/min, 15 minute gradient from 2% B to 60% B over 15 minutes. Monitoring of UV absorption was done at 220 nm or method C: A gradient of 95% A (10 mM ammonium acetate) to 95% B (10% 10 mM ammonium acetate/ 90% MeCN), flow-rate 0.8 mL/min, over 15 minutes. method D: A gradient of 95% A (10 mM ammonium acetate) to 35% B (10% 10 mM ammonium acetate/ 90% MeCN), flow-rate 0.8 mL/min, over 15 minutes. The synthesis of ligands was carried out as shown in
Compound 1. (di-tert-butyl 2,2′-(4-(3-(benzyloxy)-2-(((benzyloxy)carbonyl)amino)-3-oxopropyl)-10-((di-tert-butoxyphosphoryl)methyl)-1,4,7,10-tetraazacyclododecane-1,7-diyl)diacetate). 2-(((benzyloxy)carbonyl)amino)-3-(1,4,7,10-tetraazacyclododecan-1-yl)propanoate (1.475 g, 3.05 mmol, 1 eq) and paraformaldehyde (0.34 g, 11.45 mmol, 3.75 eq.) were dissolved in THF and stirred under N2 for 30 minutes vigorously. Tri-tert-butyl phosphite (1.145 g, 4.5 mmol, 1.5 eq.) was then added and the reaction was stirred for 16 hours. Reaction control by LCMS showed the product as [M+H]+690.4. The reaction mixture was then filtered, concentrated in vacuo, and redissolved in EtOAc (200 mL). The organic layer is washed with 50 mL saturated Na2CO3 and 50 mL brine, dried with sodium sulfate, and concentrated to afford the di-alkylated intermediate (1.73 g, 2.5 mmol, 82%), which was used in the subsequent step without further characterization. The intermediate was dissolved in MeCN (75 mL). K2CO3 (0.68 g, 4.9 mmol, 2 eq.) was added and the mixture was stirred vigorously after addition of tert-butyl bromoacetate (0.766 g, 0.575 mL, 3.94 mmol, 1.6 eq). After 16 hours, water (70 mL) was added and most of the MeCN was removed in vacuo. The oily residue was taken up in EtOAc (80 mL) and washed with water and brine (100mL each). The The organic layer was separated, dried with sodium sulfate, and concentrated to afford the crude product, which was redissolved in MeCN (5 mL) and purified using preparative HPLC (method A). The product elutes at 12.2 minutes. Fractions containing the product are pooled and lyophilized to afford the product as a white powder (0.43 g, 0.46 mmol, 20% yield). 1H-NMR (CDCl3, 500 MHz, ppm): 9.9 (s, br, 2H), 7.36-7.34 (m, 10H), 5.25-5.16(m, 4H), 4.3(m, 1H), 3.35-2.68(m, 21H), 2.04(s, 4H), 1.51-1.43(m, 36H). 13C-NMR (CDCl3, 125 MHz, ppm): 175.4, 170.6, 170.3, 135.0, 131.1, 129.4, 128.6-127.9, 81.4, 81.0, 67.7, 57.3-47.4, 30.6, 28.2, 22.1. 31P-NMR (CDCl3, 200 MHz, ppm): 17.7. LC-ESI-MS: calcd. for C47H76N5O11P4: 917.5 Found: 918.6 [M+H]+, Rt=7.8 min (method C).
Compound 2. 2-amino-3-(4,10-bis(2-(tert-butoxy)-2-oxoethyl)-7-((di-tert-butoxyphosphorryl)methyl)-1,4,7,10-tetraazacyclododecan-1-Apropanoic acid. Compound 1 of
Compound 3. 2,2′-(4-(2-amino-2-carboxyethyl)-10-(phosphonomethyl)-1,4,7,10-tetra-azacyclododecane-1,7-diyl)diacetic acid. 1H-NMR (CD3OD, 500 MHz, ppm): 5.15 (s, br, 2H), 4.26-2.46 (m, 25H). 13C-NMR (CD3OD, 125 MHz, ppm): 175.1, 169.6, 55.4-48.9. 31P-NMR (CD3OD, 200 MHz, ppm): 6.3. LC-ESI-MS: calcd. for C16H32N5O9P: 469.2 Found: 470.1 [M+H]+, Rt=1.1 min (method B).
Compound 4a. 2,2′-(4-(2-carboxy-2-(2-phenylpropanamido)ethyl)-10-(phosphonomethyl)-1,4,7,10-tetraazacyclododecane-1,7-diyl)diacetic acid. 1H-NMR (CD3OD, 500 MHz, ppm): 7.38-7.23 (m, 5H), 5.15 (s, br, 1H), 4.76 (m, 1H), 3.98-2.86 (m, 24H), 1.47 (t, 3H). 13C-NMR (CD3OD, 125 MHz, ppm): 159.4, 140.9, 128.5-126.8, 116.7, 114.4, 53.3, 49.5, 46.1, 17.1. 31P-NMR (CD3OD, 200 MHz, ppm): 0.8. LC-ESI-MS: calcd. for C25H40N5O10P: 601.2 Found: 602.4 [M+H]+, Rt=2.1 min (method B).
Compound 4b. 2,2′-(4-(2-carboxy-2-((1-(4-isobutylphenyl)ethyl)amino)ethyl)-10-(phosphonomethyl)-1,4,7,10-tetraazacyclododecane-1,7-diyl)diacetic acid. 1H-NMR (CD3OD, 500 MHz, ppm): 7.28 (d, 2H), 7.20 (d, 2H), 4.73 (s, br, 1H), 3.91-2.87 (m, 24H), 2.45(m, 2H), 1.84(m, 1H), 1.48-1.29(m, 5H), 0.89 (d, 6H). 13C-NMR (CD3OD, 125 MHz, ppm): 172.8, 169.6, 140.5, 138.2, 129.5, 127.0, 54.7-48.2, 30.0, 21.3, 17.1. 31P-NMR (CD3OD, 200 MHz, ppm): 3.5. LC-ESI-MS: calcd. for C28H48N5O9P: 629.3 Found: 630.5 [M+H]+, Rt=5.6 min (method B).
Compound 5a. 2,2′-(4-(2-carboxy-2-(2-(4-isobutylphenyl)propanamido)ethyl)-10-(phosphonomethyl)-1,4,7,10-tetraazacyclododecane-1,7-diyl)diacetic acid. 1H-NMR (CD3OD, 500 MHz, ppm): 7.39-7.26 (m, 5H), 5.02 (s, br, 1H), 4.15-2.46 (m, 24H), 1.64-1.46 (m, 3H). 13C-NMR (CD3OD, 125 MHz, ppm): 169.7, 166.3, 160.4, 142.7, 128.5-125.8, 119.6, 117.3, 114.9, 112.7, 53.3-49.5, 27.2, 20.85. 31P-NMR (CD3OD, 200 MHz, ppm): 6.1. LC-ESI-MS: calcd. for C24 H41 N6O8; P: 573.3 Found: 574.6 [M+H]+, Rt=2.3 min (method B).
Compound 5b. 2, 2′-(4-(2-amino-3-((2-(2-(4-isobutylphenyl)propanamido)ethyl)amino)-3-oxopropyl)-10-(phosphonomethyl)-1,4,7,10-tetraazacyclododecane-1,7-diyl)diacetic acid. 1H-NMR (CD3OD, 500 MHz, ppm): 7.24 (d, 2H), 7.10 (d, 2H), 4.21-2.44 (m, 30H), 1.94-1.29(m, 6H), 0.89 (d, 6H). 13C-NMR (CD3OD, 125 MHz, ppm): 175.4, 169.8, 167.4, 140.2, 138.8, 128.9, 126.7, 54.6-48.1, 38.2, 30.1, 21.3, 17.1. 31P-NMR (CD3OD, 200 MHz, ppm): 6.1. LC-ESI-MS: calcd. for O31l H54N7O9P: 699.3 Found: 700.6 [M+H]+, Rt=6.8 min (method B).
Lanthanide complexes. MS (ESI+): m/z [Gd(3)] calcd. for C16H28GdN5O9P: 623.1 Found 625.1 [M+2H]+. [Eu(3)] C16H28EuN5O9P calcd. 618.1 Found 620.1 [M+2H]+. [Gd(4a)] calcd. for C24H36GdN5O9P: 755.1 Found 757.2 [M+2H]+. [Eu(4a)] C24H36EuN5O9P calcd. 750.1 Found 752.1 [M+2H]+. [Gd(4b)] calcd. for C29H44GdN5O10P: 811.2 Found 813.4 [M+2H]+. [Eu(4b)] C29H44EuN5O10P calcd. 806.2 Found 808.4 [M+2H]+. [Tb(4b)] C29H44TbN5O10P calcd. 812.2 Found 814.4 [M+2H]+. [Gd(5a)] calcd. for C24H38GdN6O8P: 727.2 Found 728.2 [M+H]+. [Eu(5a)] C24H38EuN6O8P calcd. 722.2 Found 723.3 [M+H]+. [Gd(5b)] calcd. for C31H51GdN7O9P: 852.3 Found 853.5 [M+H]+. [Eu(5b)] C31H51EuN7O9P calcd. 849.3 Found 850.4 [M+H]+.
Relaxivity. Longitudinal relaxation times T1, were measured on Bruker Minispecs mq20 (20 MHz) and mq60 (60 MHz) using an inversion recovery method with 10 inversion time values ranging from 0.05×T1 to 5×Ti. Relaxivity was calculated from a linear plot of 3 different concentrations ranging from 0.25 to 1.0 mM versus the corresponding inverse relaxation times. The temperature was adjusted to 37° C. See Table 9.
Luminescence lifetime measurements of Eu complexes in H2O and D2O were performed on a Hitachi f-4500 fluorescence spectrophotometer. Concentrations of the samples were 5-10 mM. For the measurements in D2O, the complexes were first dissolved in D2O (99.98% D), lyophilized, and dissolved in D2O again to reduce the amount of residual H2O. Measurements were taken with the following settings: Excitation at 396 nm, emission at 616 nm, 30 replicates, 0.04 ms temporal resolution (0-20 ms), PMT voltage=400 V. Lifetimes were obtained from monoexponential fits of the data using Igor Pro software. See Table 10
17O NMR of Gd complexes for Determination of TM. 17O NMR measurements of solutions were performed at 11.7 T on 350 μL samples contained in 5 mm standard NMR tubes on a Varian spectrometer. Temperature was regulated by air flow controlled by a Varian VT unit. 17O transverse relaxation times of a >4 mM solution of Gd(3) and Gd(5a) (pH 7.4, 10 mM PBS buffer) were measured using a CPMG sequence. The concentration of the sample was determined by ICP-MS. Reduced relaxation rates, 1/T2r were calculated from the difference of 1/T2 between the sample and the water blank, and then divided by the mole fraction of coordinated water. The temperature dependence of 1/T2r was fit to a 4-parameter model as previously described. The Gd—O hyperfine-coupling constant, A/h, was assumed to be 3.8×106 rad/s, the Gd—O distance was assumed to be 3.1 Å. See
310kex × 106 (s−1)
310 TM (ns)
Synthesis of modified trimer Gd3L3-COOH.: HOOC—(CH2)CONH—Gd(DOTAla)-G-Gd(DOTAla)-G-Gd(DOTAla)-GPC(Acm)-CONH2. Manual solid phase peptide synthesis was carried out as previously reported (Boros et al, J. Am. Chem. Soc., 2012, 134, 19858-68). The originally synthesized sequence for H9L3 was altered to HOOC—(CH2)CONH-DOTAla-G-DOTAla-G-DOTAla-GPC(Acm)-CONH2 by following the last DOTAla coupling step with N-terminal capping with succinic anhydride (10 eq succinic anhydride, 10 eq DIPEA, 2 hours). The peptide was then subsequently cleaved from the solid support using the cleavage-cocktail (TFA/DDT/TIPS/Water (9.25:0.25:0.25:0.25)). The resin was filtered off and the filtrate concentrated with a gentle nitrogen flow, and resuspended in cold Et2O. The crude peptide was isolated, lyophilized and complexed with Gd. The trimeric metal complex was then purified using preparative HPLC (column: 250 mm Phenomenex C18, A, 10mM ammonium acetate in water; B, 10% 10 mM ammonium acetate in water with 90% MeCN, flow-rate 15 mL/min, from 5% B to 35% B over 15 minutes, HPLC, method D: Rt=6.6 min, MS-ESI: m/z: 1160.3 (calcd. 1160.5) [M+2H]2+.
Animal model for imaging with Gadovist/ Gd3L3-COOH: All experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Female nude mice (nu/nu), 4-6 weeks of age, were used in this study. For intracranial implantation, 105 human U87 cells suspended in 5 μL of sterile PBS were injected into the right frontal hemisphere of all the animals using a stereotactic fixation device. Tumor growth was staged every 5 days using T2 weighted images. After 17 days, the tumors were found to have reached a suitable size for imaging and quantification.
Imaging protocol Gadovist/ Gd3L3-COOH: Animals were anesthetized with isoflurane (1-2%) and placed in a specially designed cradle with body temperature maintained at 37° C. The tail vein was cannulated for intravenous (i.v.) delivery of the contrast agent while the animal was positioned in the scanner. Imaging was performed at 4.7 T, using a custom-built transmit-receive surface coil to acquire Dynamic Contrast enhanced (DCE) MRI images and T1-weighted images. Doses of the contrast agents were adjusted to 150 μmol/kg (Gadovist/ Gd3L3-COOH, on a per Gd basis). T1-weighted images were acquired using a Fast Low Angle Shot (FLASH) sequence. T1 and T2 weighted images were acquired before and after the DCE image acquisition. Image acquisition parameters were: TE/TR=2.5/83 ms, averages=16, field-of-view=2.0 cm, image matrix=144×144 (in-plane resolution=139 m), 0.5 mm slice thickness, 9 image slices. The DCE sequence consisted of a T1-weighted gradient-echo sequence with TE=2.1 ms, TR=25 ms, 2 averages, flip angle=60°, FOV=2.0 cm, matrix=72×72 (in-plane resolution=278 μm), 0.5 mm slice thickness, 1 image slice, 90 repetitions, temporal resolution=3.6 s. The contrast agent was injected approximately 1 minute after commencement of the DCE imaging sequence using an intravenous tail vein catheter. The signal intensity in the tumor region of interest (ROI) was analyzed using an in-house written MATLAB program, which models the tumor signal enhancement using the two-compartment model (Tofts et al, Magn. Reson. Med., 1991 17, 357-367; Tofts et al., J. Magn. Reson. Imaging, 1997, 7, 91-101, 3; Tofts et al., J Magn. Reson. Imaging 1999, 10: 223-232), to extract the volume fraction of the extra-vascular extra-cellular (EES) space (ve), the volume transfer constant between the plasma and EES (Ktrans), and the rate constant between the EES and the blood plasma (kep). Briefly, the time dependence of the tumor signal intensity is fit to equation 1.
where R1(t) is the longitudinal relaxation rate, a is the flip angle, and TR is the repetition time. R1(t) depends on the contrast agent relaxivity (r1), the pre-contrast longitudinal relaxation rate (R1(0)), and the tissue concentration of the contrast agent tracer (Ct(t)) as described by equation 2.
R1(t)=R1(0)+r1*Ct(t) [Eq. 2]
In turn, Ct(t) is derived from the arterial input function (AIF), Cp(t), as described by equation 3.
The AIF is modeled as a bi-exponential function with parameters a1 and k1 describing the fast equilibration between the plasma and extracellular space, a2 and k2 describing the clearance of contrast agent by the kidneys, and D is the contrast agent dose (mmol Gd/kg bodyweight) administered by intravenous injection. We have used the AIF parameters determined empirically (McGrath et al., Magn. Reson. Med., 2009, 61, 1173-1184). See
Probe preparation MS-325/Gd(9)/Gd(4a): Gd(9) and Gd(4a) were prepared according to the procedure of Example 3 as described above. MS-325 was obtained from a commercial source as a 0.1M solution (Gadofosveset, trade names Vasovist, Ablavar). All stock solutions were diluted with PBS to obtain a 40 mM probe concentration, which was determined by ICP-MS.
Animal model for blood pool imaging with MS-325/Gd(9)/Gd(4a): All experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Healthy female nude mice (nu/nu), 4-6 weeks of age were used in this study.
Animal model for liver imaging with Gd(9): Strain A/J male mice (Jackson Laboratories, Bar Harbor, Me., USA) were administered 0.1 ml of a 40% solution of carbon tetrachloride (CCl4; Sigma) in olive oil by oral gavage, three times a week for 20 weeks (n=8); age matched controls received only pure olive oil (n=4), or no vehicle (n=6). Animals from both models were imaged one week after the last injection to avoid acute effects of DEN or CCl4.
Imaging protocol MS-325/Gd(9)/Gd(4a): Animals were anesthetized with isoflurane (1-2%) and placed in a specially designed cradle with body temperature maintained at 37° C. The tail vein was cannulated for intravenous (i.v.) delivery of the contrast agent while the animal was positioned in the scanner. Imaging was performed at 4.7 T, using a custom-built volume coil to acquire T1-weighted images. The animal was positioned such that the major organs (heart, stomach, liver, intestines, kidney) were visible in the field of view. Doses of the contrast agents were adjusted to 100 μmol/kg. T1-weighted images were acquired using a 3D Fast Low Angle Shot (FLASH) sequence. MS-325/Gd(4a): FA: 40. Resolution: 0.25 mm/pxl isotropic, Averages: 1, FOV: 4.8 cm×2.4 cm×2.4 cm, Matrix Size: 192×96×96, TE: 1.54, TR: 15.29), with one gated and one non-gated image acquired pre-injection, followed by 5 non-gated 3D FLASH scans, which then were followed by 7 gated and 7 non-gated 3D flash scans up to 1h post injection. Gd(9) healthy animals: FA: 20. Resolution: 0.375mm/pxl isotropic, Averages: 1, FOV: 4.8 cm×2.4 cm×2.4 cm, Matrix Size: 128×64×64, TE: 1.35, TR: 11.01. Gd(9) CCl4 mice: FA: 20. Resolution: 0.25 mm/pxl isotropic, Averages: 1, FOV: 4.8 cm×2.4 cm×2.4 cm, Matrix Size: 192×96×96, TE: 1.54, TR: 15.29.
Although the present invention has been described in detail with reference to certain embodiments, one skilled in the art will appreciate that the present invention can be practiced by other than the described embodiments, which have been presented for purposes of illustration and not of limitation. Therefore, the scope of the appended claims should not be limited to the description of the embodiments contained herein.
This application claims priority from U.S. Patent Application No. 61/725,339 filed Nov. 12, 2012 and U.S. Patent Application No. 61/858,002 filed Jul. 24, 2013.
This invention was made with government support under grant number EB 009062 awarded by the National Institute of Biomedical Imaging and Bioengineering. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2013/069701 | 11/12/2013 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61725339 | Nov 2012 | US | |
| 61858002 | Jul 2013 | US |
