Patent Application
20080085237
The novel compounds and methodologies according to the present invention, which incorporating weakly basic moieties (pKa about 8 or greater) into 2-nitroimidazole hypoxia markers labeled with, e.g., a halogen, a positron-emitting radionuclide, or a non-metal, facilitate the non-invasive analysis of normal tissue hypoxia and the analysis of changes in tissue hypoxia. In particular, the invention provides convenient techniques for measuring hypoxia prior to therapeutic intervention that, in turn, allow for the selection of patients for hypoxia-based therapeutic interventions in an effective and timely manner. The invention also provides a way for following the effectiveness of hypoxia-based, therapeutic interventions in diseased normal and malignant tissue. In particular, the compounds of the invention are useful for detecting hypoxic conditions present in, e.g., tissues of the brain, lungs, heart, eyes, kidney, liver, pancreas, thymus, intestines, urogenital organs, stomach, and bone. The hypoxic conditions can result from ischemia (e.g., as a result of stroke), inflammation, wound healing, and cancer.
The invention provides compounds for the non-invasive detection of both chronic and acute hypoxia using PET, MRS, and MRI. The compounds efficiently detect chronic and acute hypoxia. The compounds of the invention provide increased signal-to-noise ratios and have the ability to detect acute or fluctuating hypoxia with greater sensitivity than prior art markers. The ability to detect acute hypoxia is important because it is widely believed among cancer biologists that acute hypoxia has an inordinate impact on cancer therapy.
Compounds of the invention include fluorinated 2-nitroimidazole derivatives for the non-invasive detection of hypoxia in diseased normal and malignant tissue by means of [18F]PET, [19F]MRS, or [19F]MRI. The 2-nitroimidazole moiety of the compounds undergoes bioreduction to intermediates that bind covalently to peptides and proteins in tissue cells that have a pO2≦10 mmHg, such that stable adducts are formed that act as markers for tissue hypoxia. The introduction of [18F] (and/or other positron emitting radionuclides) or [19F] (and/or other non-radioactive halogens or non-metals) into the 2-nitroimidazole allows for the detection of tissue hypoxia by means of physically non-invasive [18F]PET, [19F]MRS, or [19F]MRI. The compounds of the invention can be used in the non-invasive study of hypoxia in diseased normal tissue (e.g., arthritic tissue) and malignant tissue (e.g., cancer tissue).
The invention can be used in two ways. First, the level of tissue hypoxia prior to therapy can be assessed allowing for selection of patients who may benefit from a hypoxia-based intervention. Second, changes in tissue hypoxia in response to therapeutic interventions, such as ionizing radiation, hyperthermia, hypoxic cell radiosensitizers, bioreductive cytotoxins, anti-inflammatory agents, or growth factor inhibitors can be followed as a measure of the success of the intervention.
The compounds of the invention constitute an improvement over prior art compounds in several respects, not the least of which include the following. 1) The compounds of the invention reduce background “noise” typically observed with existing PET, MRS, and MRI reagents when used in the non-invasive measurement of hypoxia. 2) Acid salts of 2-nitroimidazole compounds possessing a weakly basic substituent are water-soluble, thereby facilitating administration in both human and experimental animal applications. 3) Weakly basic reagents have much shorter plasma half-lives in humans. For example, pimonidazole has a much shorter plasma half-life (t1/2=5.1±0.8 hours) than hypoxia markers such as misonidazole (t1/2=9.3 hours) or EF5 (t1/2=11.7±2.7 hours). Therefore, unmetabolized, weakly basic markers will be cleared much more rapidly from circulation thereby increasing signal-to-noise in, e.g., [18F]PET, [19F]MRS, and [19F]MRI] analyses. 4) Selective uptake of weakly basic PET reagents into tissue cells above extracellular concentrations will increase the rate of binding to hypoxic cells and enhance sensitivity of detection. The methodology of the present invention recognizes that enhanced uptake is the result of differentials between intracellular and extracellular pH in cells of solid tumors. 5) Conjugate bases of compounds of the invention possessing weakly basic substituents (e.g., [18F]PET, [19F]MRS, and [19F]MRI reagents of the invention) have intermediate octanol-water partition coefficients. This means that the compounds readily penetrate all tissues including brain where they are concentrated about 3 fold above plasma levels. Therefore, weakly basic PET, MRS, and MRI compounds of the invention can be used for investigating hypoxia in all normal and tumor tissues, whereas hydrophilic markers in the prior art are effectively excluded from many normal tissues of interest. It is known that central nervous system toxicity is limiting for weakly basic 2-nitroimidazole hypoxia markers. However, PET compounds are used in trace amounts and central nervous system toxicity is not a significant issue. Even relatively high doses (0.5 g/m2; 750-1000 mg/patient; 50% of the maximally tolerated single dose) of the weakly basic hypoxia marker, pimonidazole, has been used clinically with an extremely low frequency of even the mildest of central nervous system (CNS) effects, such as a sensation of warmth, indicating that CNS toxicity would not prevent the use of higher concentrations of the weakly basic 2-nitroimidazole compounds of the present invention for MRS or MRI (e.g., the use of [19F]-fluorinated, weakly basic 2-nitroimidazole compound of the present invention for [19F]MRS or [19F]MRI). 6) Adducts of hypoxia markers with weakly basic substituents are more stable than hypoxia markers that lack a weakly basic substituent. This has the effect of stabilizing the hypoxia signal over currently available PET, MRS, and MRI markers of the prior art (in particular, the fluorinated hypoxia markers of the present invention demonstrate a hypoxia signal that is more stable than the [18F]PET, [19F]MRS, and [19F]MRI hypoxia markers of the prior art). 7) The weakly basic compounds of the present invention permit detection of acute hypoxia with much higher sensitivity than PET, MRS, and MRS markers of the prior art. The weakly basic substituent of the compounds of the invention promotes their concentration in cells experiencing fluctuating hypoxia in a high extracellular pH (pHe) tissue microenvironment. This occurs due to differentials in intracellular and extracellular pH of cells experiencing fluctuating hypoxia; pH gradients exist in solid tissues such that cells experiencing fluctuating hypoxia are at relatively high pH.
The novel compounds provided herein are those defined by the structural formulas (I) and (II).
R1 can be selected from a halogen (e.g., fluorine (F), chlorine (Cl), bromine (Br), iodine (I), or astatine (At)), a positron emitting radionuclide (e.g., [11C], [13N], [15O], [18F], [52Fe], [55Co], [61Cu], [62Cu], [64Cu], [62Zn], [63Zn], [70As], [71As], [74As], [76Br], [79Br], [82Rb], [86Y][89Zr], [110In], [120I], [124I], [122Xe], [94mTc], [94Tc], or [99mTc]), a non-metal, a lower alkyl substituted to contain a halogen, a lower alkyl substituted to contain a positron emitting radionuclide, a lower alkyl substituted to contain a non-metal, a tosylate, a mesylate, a tryflate, a hydrogen, or a hydroxyl; and R2 and R3 can be independently selected from a lower alkyl or a hydroxyalkyl, or are linked to form a five-, six-, or seven-membered heterocyclic ring containing at least one nitrogen atom (e.g., at least 2, 3, or 4 nitrogen atoms); with the caveat that if R1 is hydrogen or hydroxyl, at least one of R2, R3, or the heterocylic ring contains a halogen, a positron emitting radionuclide, a non-metal, a lower alkyl substituted to contain a halogen, a lower alkyl substituted to contain a positron emitting radionuclide, a lower alkyl substituted to contain a non-metal, a tosylate, a mesylate, or a tryflate.
Preferably, R2 and R3 are linked to form a five-, six-, or seven-membered heterocycyclic ring that has at least one nitrogen atom, but excludes groups in the ring that decrease basicity, such as O, S, or N-acyl. At least one N atom in structure (I) may be in salt form with anionic counterions including, but not limited to, halide. In the case of multiple N atoms, at least one N may be substituted with a lower alkyl, hydroxyalkyl or fluoroalkyl group. Further, R2 and R3 may be substituted at carbon with a moiety independently selected from the group of hydrogen, tosylate, mesylate, tryflate, [19F]fluorine or [18F]fluorine.
Examples of preferred compounds within this group are as shown in Chart A:
The compounds defined by structural formulas (VI-XVII), including salts thereof, are useful for detecting hypoxia in diseased normal and malignant tissue either alone or in conjunction with other PET markers of tissue physiology (e.g., [18F]-fluorodeoxyglucose, [18F]-FDG).
Radiolabeled compounds of the invention are useful compositions for imaging, detection, and diagnosis of disease in a subject. Numerous radiolabels may be used to generate radiolabeled compounds that are useful in imaging and detection. For example, a non-limiting list of radiolabels that may be used to generate radiolabeled compounds include 11C, 13N, 15O, 18F, 52Fe, 62Cu, 64Cu, 67Cu, 67Ga, 68Ga, 76Br, 86Y, 89Zr, 94mTc, 94Tc, 99mTc, 111In, 123I, 124I, 125I, 131I, 154-158Gd, and 175Lu. Particularly preferred radiolabels comprise, or alternatively consist of, 18F, 64Cu, 76Br, I124 and mixtures thereof.
As an example, 18F can be obtained from cyclotrons after bombardment of 18O-enriched water with protons. The enriched water containing H-18F can be neutralized with a base having a counter-cation that is any alkylammonium, tetraalkylammonium, alkylphophosphonium, alkylquanidium, alkylamidinium, or alkali metal (M), such as potassium, cesium, or other monovalent ions that are strongly chelated to a ligand such as Kryptofix 222 (4,7,13,16,21,24-hexaoxa-1,10-diazabycyclo[8.8.8]hexacosane), such that the resulting alkali metal-ligand complex is freely soluble in organic solvents such as acetonitrile, dimethylsulfoxide, or dimethylformamide. The water can be evaporated off to produce a residue of countercation-18F, which can be taken up in an organic solvent for further use. In general, the counter-cation is selected to enable the fluoride ion to react rapidly in an organic phase with a halogen.
Because fluoride is the most electronegative element, it has a tendency to become hydrated and lose its nucleophilic character. To minimize this, the labeling reaction preferably is performed under anhydrous conditions. For example, fluoride (as potassium fluoride or as a complex with any of the other counter-ions discussed above) can be placed in organic solvents, such as acetonitrile or THF. With the assistance of agents that bind to the counter-cation, such as Kryptofix 2.2.2 (4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]-hexacosane), the fluoride ion becomes very nucleophilic in these solvents. The remaining portion of the chelate molecule of the invention then can be added to the solvent and the chelate thereby labeled with the 18F.
Although potassium is useful as the metal in the counter-cations in accordance with the present invention, cesium may be preferred to potassium because cesium is a larger ion with a more diffuse charge. Accordingly, cesium has looser ionic interactions with the small fluoride atom, and therefore does not interfere with the nucleophilic properties of the fluoride ion. For similar reasons, potassium may be preferred to sodium, and, in general, the suitability of a lanthanide metal as the metal in the counter-cation in accordance with the present invention increases as you go down the periodic table. Group Ib reagents, such as silver, also are useful as counter-ions in accordance with the present invention. Further, organic phase transfer-type ions, such as tetraalkylammonium salts, also can be used as counter-cations.
The compounds of the invention can be used to preferentially target tumor tissue. Compounds of the invention may be administered to a mammalian subject, such as a human, directly or in combination with any pharmaceutically acceptable carrier or salt known in the art. Pharmaceutically acceptable salts may include non-toxic acid addition salts or metal complexes that are commonly used in the pharmaceutical industry. Examples of acid addition salts include organic acids such as acetic, lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic, palmitic, suberic, salicylic, tartaric, methanesulfonic, toluenesulfonic, or trifluoroacetic acids or the like; polymeric acids such as tannic acid, carboxymethyl cellulose, or the like; and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid phosphoric acid, or the like. Metal complexes include zinc, iron, and the like. One exemplary pharmaceutically acceptable carrier is physiological saline. Other physiologically acceptable carriers and their formulations are known to one skilled in the art and described, for example, in Remington's Pharmaceutical Sciences, (18th edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, Pa. Pharmaceutical formulations of a therapeutically effective amount of a compound of the invention, or pharmaceutically acceptable salt-thereof, can be administered orally, parenterally (e.g., intramuscular, intraperitoneal, intravenous, or subcutaneous injection, inhalation, intradermally, optical drops, or implant), nasally, vaginally, rectally, sublingually, or topically, in admixture with a pharmaceutically acceptable carrier adapted for the route of administration.
Methods well known in the art for making formulations are found, for example, in Remington's Pharmaceutical Sciences (18th edition), ed. A. Gennaro, 1990, Mack Publishing Company, Easton, Pa. Compositions intended for oral use may be prepared in solid or liquid forms according to any method known to the art for the manufacture of pharmaceutical compositions. The compositions may optionally contain sweetening, flavoring, coloring, perfuming, and/or preserving agents in order to provide a more palatable preparation. Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid forms, the active compound is admixed with at least one inert pharmaceutically acceptable carrier or excipient. These may include, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, sucrose, starch, calcium phosphate, sodium phosphate, or kaolin. Binding agents, buffering agents, and/or lubricating agents (e.g., magnesium stearate) may also be used. Tablets and pills can additionally be prepared with enteric coatings.
Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and soft gelatin capsules. These forms contain inert diluents commonly used in the art, such as water or an oil medium. Besides such inert diluents, compositions can also include adjuvants, such as wetting agents, emulsifying agents, and suspending agents.
Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of suitable vehicles include propylene glycol, polyethylene glycol, vegetable oils, gelatin, hydrogenated naphalenes, and injectable organic esters, such as ethyl oleate. Such formulations may also contain adjuvants, such as preserving, wetting, emulsifying, and dispersing agents. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for the peptide agents of the invention include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
Liquid formulations can be sterilized by, for example, filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, or by irradiating or heating the compositions. Alternatively, they can also be manufactured in the form of sterile, solid compositions which can be dissolved in sterile water or some other sterile injectable medium immediately before use.
Compositions for rectal or vaginal administration are preferably suppositories which may contain, in addition to active substances, excipients such as coca butter or a suppository wax. Compositions for nasal or sublingual administration are also prepared with standard excipients known in the art. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops or spray, or as a gel.
The amount of active ingredient in the compositions of the invention can be varied. One skilled in the art will appreciate that the exact individual dosages may be adjusted somewhat depending upon a variety of factors, including the chemical compound being administered, the time of administration, the route of administration, the nature of the formulation, the rate of excretion, the nature of the subject's conditions, and the age, weight, health, and gender of the patient. In addition, the severity of the condition targeted by a compound of the invention will also have an impact on the dosage level. Generally, dosage levels of between 0.1 μg/kg to 100 mg/kg of body weight are administered daily as a single dose or divided into multiple doses. Preferably, the general dosage range is between 250 μg/kg to 5.0 mg/kg of body weight per day. Wide variations in the needed dosage are to be expected in view of the differing efficiencies of the various routes of administration. For instance, oral administration generally would be expected to require higher dosage levels than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, which are well known in the art. In general, the precise therapeutically effective dosage will be determined by the attending physician in consideration of the above identified factors.
The compounds of the invention may be prepared in high yield using simple straightforward methods as exemplified by the examples below. It is to be understood that while the invention has been described in conjunction with the preferred specific embodiments thereof, that the foregoing description as well as the examples that follow are intended to illustrate and not limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.
The following examples are provided so that those of ordinary skill in the art can see how to make and use the compounds of the invention. The examples are not intended to limit the scope of what the inventors regard as their invention. All starting materials and reagents are commercially available.
The starting material (1) (460 mg, 1.8 mmol) was dissolved in 50 ml THF and Et3N (0.5 ml) was added and followed by dropwise addition of MsCl (0.28 ml, 3.6 mmol). The reaction was stirred at room temperature for 20 min. Thin layer chromatography (TLC) showed the starting material was almost completely reacted. After the workup the crude reaction product was purified by column chromatograph (EtOAc) to afford 500 mg of desired mesylated product (2) (yield is 84%). 1HNMR(DMSO-d6, δppm): 1.63 (m, 2H, CH2), 1.90 (m, 4H, 2xCH2), 2.10 (m, 2H, CH2), 2.24 (t, 2H, CH2, J=6.9 Hz), 2.48 (m, 2H, CH2), 3.15 (s, 3H, CH3), 4.41 (t, 2H, CH2, J=6.9 Hz), 4.60 (m, 1H, CH), 7.15 (d, 1H, CH═, J=1.2 Hz), 7.65 (d, 1H, CH═, J=1.2 Hz).
Kryptofix 222 (270 mg, 0.72 mmol) was dissolved in 5 ml of acetonitrile (CH3CN). To this solution was added anhydrous powder potassium fluoride (99.99+%, 33 mg, 0.57 mmol) followed by mesylate (2) (80 mg, 0.24 mmol). The resulting mixture was refluxed for 2 hrs in an oil bath (95-100° C.). After workup, the crude reaction product was purified by preparative TLC to give 31 mg of fluoride (3, X). The overall yield was 50%. For Compound (3,X): 1HNMR(DMSO-d6, δppm): 1.67 (m, 2H, CH2), 1.92 (m, 4H, 2xCH2), 2.07 (m, 2H, CH2), 2.23 (t, 2H, CH2, J=6.6 Hz), 2.53 (m, 2H, CH2), 4.13 (brs, 1H, CH), 4.41 (t, 2H, CH2, J=6.9 Hz), 7.15 (s, 1H, imidazole), 7.64 (s, 1H, imidazole). 13C NMR (DMSO-d6, δppm): 27.34, 35.87, 48.32, 51.40, 54.83, 58.84, 128.41, and 128.58.
The starting material (4) (4 g, 14.85 mmol) was dissolved in 200 ml THF. To this solution was added Et3N (5 ml) followed by dropwise addition of MsCl (3 ml, 38.8 mmol). The reaction was stirred at room temperature for 30 min. TLC showed that the starting material was almost all consumed. After the workup, the crude reaction mixture was purified by flash column chromatograph (EtOAc) to afford 3.1 g of di-mesylated product (5), yield is 49.1%. 1HNMR(DMSO-d6, δppm): 1.71 (m, 2H, CH2), 1.93 (m, 2H, CH2), 2.47 (m, 2H, CH2), 2.68 (m, 4H, 2x CH2), 3.05 (s, 3H, CH3SO—), 3.16 (s, 3H, CH3SO—), 4.55 (dd, 1H, imidazole-CHa—, J=14.4 Hz, 8.7 Hz), 4.66 (m, 1H, O—CH), 4.84 (dd, 1H, imidazole-CHa—, J=14.4 Hz, 8.7 Hz), 5.00 (m, 1H, O—CH), 7.16 (d, 1H, imidazole, J=0.9 Hz), 7.59 (d, 1H, imidazole, J=0.9 Hz).
Kryptofix 222 (MW 376.5, 42 mg, 0.112 mmol) was dissolved in 2 ml of acetonitrile (CH3CN). To this solution was added anhydrous potassium fluoride (99.99+%, 22 mg, 0.379 mmol) followed by di-mesylate (5) (28 mg, 0.0658 mmol). After the di-mesylate (5) was all dissolved, the mixture was refluxed for 30 min. After the workup, the crude reaction mixture was purified by preparative TLC to give 20 mg (87% yield) of mono-fluorine exchanged compound (6). For compound (6): 1HNMR(DMSO-d6, δppm): 1.68 (m, 2H, CH2), 1.90 (m, 2H, CH2), 2.43 (m, 2H, CH2), 2.62 (m, 2H, CH2), 2.74-2.89 (m, 2H, piperidine-CH2), 3.11 (s, 3H, CH3SO—), 4.36-4.51 (m, 1H, imidazole-CH), 4.68-4.90 (m, 2H, imidazole-CH, F—H), 5.06 (m, 1H, O—CH), 7.12 (d, 1H, imidazole, J=0.9 Hz), 7.19 (d, 1H, imidazole, J=0.9 Hz). 13C NMR(DMSO-d6, δppm): 25.89, 32.06, 50.21 (d, JF—C=20.44 Hz), 55.32, 60.15 (d, JF—C=20.81 Hz), 65.89, 89.63 (d, JF—C=169.68 Hz), 127.55, 128.46.
Pimonidazole (3.7 g, 14.5 mmol), p-toluenesulfonic anhydride (5.7 g, 17.5 mmol) and DMAP (1.78 g, 14.5 mmol) were added to 100 ml of anhydrous CH2Cl2 at 0° C. in an ice-water bath. After stirring for 30 min, the reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with water, dried over anhydrous sodium sulfate, filtered and concentrated. The crude reaction product was purified by column chromatography (EtOAc-Hexane=1:1) to afford 4.1 g (70% yield) of the desired tosylate (7, III). For compound (7, III): 1H NMR (CDCl3, δppm): 1.41(m, 2H, CH2), 1.52 (m, 4H, 2xCH2), 2.33-2.67 (m, 6H, 3xCH2), 2.44 (s, 3H, CH3), 4.29 (dd, 1H, imidazole-CHa—, J=14.4 Hz, 8.7 Hz), 4.87 (m, 1H, O—CH—), 4.97 (dd, 1H, imidazole-CHb—, J=14.7 Hz, J=2.7 Hz), 7.03 (d, 2H, Benzene, J=8.4 Hz), 7.25 (d, 1H, imidazole, J=0.6 Hz), 7.26 (d, 1H, imidazole, J=0.6 Hz), 7.54 (d, 2H, Benzene, J=8.4 Hz). 13C NMR(CDCl3, δppm): 21.87, 24.17, 26.10, 52.08, 55.58, 60.04, 77.42, 127.59, 127.67, 128.38, 130.22, 132.38, 145.82.
Kryptofix 222 (MW 376.5, 824 mg, 2.19 mmol) was dissolved in 6ml of acetonitrile (CH3CN). To this solution was added anhydrous potassium fluoride (99.99+%, 128 mg, 2.19 mmol) followed by tosylate (7, III) (300 mg, 0.73 mmol). After the tosylate was totally dissolved, the reaction mixture was refluxed for 2 hrs in an oil bath at 95° C. After the workup, the crude reaction product was purified by column chromatography (EtOAc-Hexane=1:1) to afford 600 mg (32% yield) of target fluorinated product (8, VI), along with 550 mg (29.4% yield) of by-product (9, VII).
For compound (8, VI), 1H NMR (CDCl3, δppm): 1.37 (m, 2H, CH2), 1.52 (m, 4H, 2xCH2), 2.39 (m, 4H, 2xCH2), 2.49-2.66 (m, 2H, piperidine-CH2), 4.45-4.58 (m, 1H, imidazole-CH), 4.79-5.02 (m, 2H, imidazole-CH, F—H), 7.10 (d, 1H, imidazole, J=0.9 Hz), 7.15 (d, 1H, imidazole, J=0.9 Hz). 13C NMR(CDCl3, δppm): 23.87, 25.81, 51.81 (d, JF—C=21.2 Hz), 55.32, 59.33 (d, JF—C=21.8 Hz), 90.14 (d, JF—C=174.6 Hz), 126.97, 128.22.
For compound (9, VII), 1H NMR (CDCl3, δppm): 1.40 (m, 6H, 3xCH2), 2.37 (m, 2H, piperidine ring: —N—Ha), 2.68 (m, 2H, piperidine ring: —N—He), 2.82-3.32 (m, 1H, piperidine-CH), 4.56 (ddd, 1H, F—Ha, JF—H=89.1 Hz, JH—H=10.2 Hz, 3.9 Hz), 4.49 (d, 2H, CH2, J=6.9 Hz), 4.53-4.59 (m, 1H, F—Hb), 7.08 (d, 1H, imidazole, J=0.9 Hz), 7.09 (d, 1H, imidazole, J=0.9 Hz). 13C NMR(CDCl3, δppm): 24.27, 26.28, 47.18 (d, JF—C=7.4 Hz), 50.86, 64.88 (d, JF—C=17.8 Hz), 80.79 (d, JF—C=172.5 Hz), 126.93, 127.85.
2-Nitroimidazole (1 molar equivalent) in acetone was mixed with epichlorohydrin (1.1 molar equivalent) and potassium carbonate (0.001 molar equivalent). The mixture was refluxed overnight and taken to dryness in vacuo to give 1-(2-hydroxy-3-chloropropyl)-2-nitroimidazole. 1-(2-Hydroxy-3-chloropropyl)-2-nitroimidazole is taken up in ethyl acetate and mixed with an equal volume of 10% aqueous sodium hydroxide with vigorous stirring for 1 hour at room temperature. The ethyl acetate layer was washed with water, dried over anhydrous sodium sulfate and taken to dryness to give 1-(2,3-epoxypropyl)-2-nitroimidazole. 1-(2,3-epoxypropyl)-2-nitroimidazole (1 molar equivalent) dissolved in acetone was mixed with N′-1,1,1,3,3,3-hexafluoroisopropylpiperazine (1.1 molar equivalent) and the solution refluxed overnight. The reaction solution was taken to dryness in vacuo to give 1-(2-hydroxy-3-(N′-1,1,1,3,3,3-hexafluoroisopropylpiperazino)-2-nitroimidazole (XVI) that was recrystallized from ethanol. The chemical intermediate, N′-1,1,1,3,3,3-hexafluoroisopropylpiperazine, was prepared by heating 1,1,1,3,3,3-hexafluoroisopropyl bromide (1.0 molar equivalent) and piperazine (1.1 molar equivalent) at reflux in ethanol overnight. The chemical intermediate 1,1,1,3,3,3-hexafluoroisopropyl bromide was prepared by reacting commercially available 1,1,1,3,3,3-hexafluoroisopropyl alcohol (1 molar equivalent) with phosphorus tribromide (0.33 molar equivalent) in ethyl ether overnight at room temperature.
Weakly basic 2-nitroimidazole hypoxia compounds of the invention are more sensitive detectors of hypoxia in cells at high pH than are 2-nitroimidazoles that lack a weakly basic moiety, such as CCI-103F. pH-dependent binding for weakly basic pimonidazole was compared to that for CCI-103F in Chinese hamster V79-4 lung fibroblasts under conditions of short-term anoxia. The pH range used (6.4 to 7.4) encompasses about 90% of extracellular pHs measured in human tumors. Eagle's minimum essential medium (MEM) containing 4.5 g/L glucose, but no carbonate, was warmed to 37° C. in a warm room and adjusted to pH 6.4, 6.8 and 7.4 by the addition of sodium bicarbonate under a stream of 5% CO2+95% nitrogen. Fetal bovine serum (FBS) was added to produce an Eagle's pH adjusted MEM containing 10% FBS. Attached V79-4 cells were harvested with EDTA-trypsin and diluted in 25 mL of the pH adjusted MEM at a concentration of 3×105 cells/mL. To this solution was added an amount of a stock solution of pimonidazole HCl or CCI-103F to produce a final concentration of 200 uM. The cell solution was then incubated under an atmosphere of 5% CO2+95% nitrogen with agitation for 3 hours. Cell lysates were analyzed by ELISA and the data normalized to protein content. The experiments for both markers were performed in triplicate.
The intensity of pimonidazole binding was greater than that for CCI-103F at all pH levels tested with the difference being greatest at the highest pH tested (Table 1). These data indicate that weakly basic 2-nitroimidazole hypoxia markers are superior reagents at all pH's but are particularly advantageous for the detection of hypoxia in microregions of tissues in which cells are at a relatively high pH. This is a direct result of the fact that intracellular concentrations (Ci) of weakly basic 2-nitroimidazoles increase steeply relative to extracellular concentrations (Ce) with increasing pH whereas no such effect is seen with 2-nitroimidazoles that lack a weakly basic moiety. Because regions in tumors include cells that experience fluctuating hypoxia and a relatively high pH microenvironment, the compounds of the invention, which exhibit increased binding to cells at high pH, are superior at detecting fluctuating hypoxia.
Weakly basic 2-nitroimidazole hypoxia compounds of the invention are more efficient at detecting fluctuating hypoxia than are compounds that lack a weakly basic moiety when measured in large spontaneous canine tumors analogous to those occurring in humans. The hydrochloride salt of the weakly basic hypoxia marker, pimonidazole, was given to 12 dogs at a dosage of 0.5 gm/m2 body surface area. Seven hours later, all 12 dogs received CCI-103F, a marker that lacks a weakly basic moiety. Two to four, widely separated biopsy samples were taken from viable regions in each tumor and immediately placed in cold 10% neutral buffered formalin. The specimens were fixed for 18-24 hours at 4° C. and then transferred into cold 70% ethanol and stored at 4° C. until mounted into paraffin blocks. Sections from formalin-fixed paraffin-embedded biopsy samples were immunostained for pimonidazole and CCI-103F binding using primary rabbit polyclonal antisera to pimonidazole and CCI-103F adducts respectively. Immunostained sections were exhaustively scanned at 400× by means of an Axioskop 50 microscope and Fluar objective and the percent immunostaining for pimonidazole and CCI-103F adducts was measured.
On average, immunostaining for pimonidazole binding was more extensive than that for CCI-103F (factor 1.25 by paired t test (p=0.032)), but, importantly, on a tumor-by-tumor basis, the factor ranged from 1.0 to 1.65. Furthermore, within a single tumor the extent of pimonidazole binding was similar to that for CCI-103F in some regions (
These data indicate that weakly basic, 2-nitroimidazole compounds labeled with [18F] or [19F] are more effective than prior art hypoxia markers lacking a weakly basic moiety for non-invasively detecting hypoxia in mammalian tissue.
All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth.
