Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/0402—Organic compounds carboxylic acid carriers, fatty acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• the present invention relates to novel radiolabeled fatty acid analogs having a cyclic organic substituent, where a positron or gamma-emitting label is placed at a position on a fatty acid backbone and an organic substituent is substituted at the 2,3; 3,4; 4,5; 5,6 and other sequence positions of a fatty acid backbone.
• These novel fatty acid analogs are designed to enter the tissues of interest by the same long chain fatty acid carrier mechanism as natural fatty acids, however, functional substituents in the 2,3; 3,4; 4,5; 5,6 and other sequence positions, block the catabolic pathway, thus trapping these analogs in a virtually unmodified form in the tissues of interest.
• FAs Fatty acids
• ATP adenosine triphosphate
• Uptake of free FAs by the myocardium occurs at an extraction percentage of 40% to 60% of blood content, which is proportional to perfusion.
• Transported to the heart as nonesterified FAs, as triglycerides in very low-density lipoprotein particles or in chylomicrons, or bound to serum albumin they pass along concentration gradients to the interstitiurn. Under these conditions, FAs supply as much as 70% of oxidatively metabolized substrate.
• the extraction of free FAs by the myocyte is regulated by several variables including FA chain length, the availability of other metabolic substrates, circulating levels of hormones, cardiac workload, and the presence or absence of ischemia.
• FAs behave like native FAs up to the ⁇ -oxidation step in the mitochondria, where it is sequestered for a long period of time (Livni, E. et al. 1990 Lipids 25: 238-40).
• the fate of an FA may be described as follows: FA passes from capillary blood into the interstitial space. It may "back-diffuse” to the vascular space or pass through the interstitial space, where it may become activated as acyl-coenzymeA (Co A). The activated FA can then be esterified to form triglycerides, incorporated into phospholipids or carried into the mitochondria and oxidized.
• n C-PA neutrophil-associated n C-PA
• n C-PAs labeled with n C on the carboxyl group is subject to loss of labeling during the first round of ⁇ -oxidation.
• Studies employing direct intracoronary administration of n CO 2 and direct myocardial monitoring demonstrate the evolution of ⁇ CO 2 within 30 seconds and a 50% clearance within 2-8 minutes.
• the rapid washout of the radiolabel due to ⁇ -oxidation and short sequential imaging periods imposes limitations on counting statistics.
• the second approach involves the use of "analog tracers" that enter a known metabolic pathway.
• metabolism of these tracers stop at a certain stage, leaving the radiolabel trapped in the cell in a known form.
• This concept has been applied to the study of glucose metabolism using glucose analogs such as l-[ ⁇ C]-2-deoxyglucose (2DG) and 2- [ 18 F]-fluorodeoxyglucose (2FDG).
• 2DG glucose analogs
• FDG 2- [ 18 F]-fluorodeoxyglucose
• the principle of metabolic trapping has been used successfully with 2FDG to measure in vivo regional glucose metabolic rates in humans. Investigations of the use of 2FDG for measuring myocardial glucose metabolism have been conducted. Similarly, FAs have also been widely used to measure metabolic activity in the myocardium.
• FAs tend to wash out very quickly due to ⁇ -oxidation, depending on the position of the radionuclide. Subsequently, the radiolabeled FA or metabolites can then accumulate in tissues other than the region of interest, primarily liver and lung, h radiohalogenated aliphatic fatty acids, such accumulation occurs frequently with 123 I, which migrates and is stored in the thyroid gland, and 18 F, which is stored in bone.
• Machulla et al reported that the ⁇ - terminal labeled FAs were more efficiently extracted than analogs labeled in the - position, and that DTD A had the highest uptake (Machulla, H. et al (1978) J. Nucl. Med. 19: 298-302).
• radiolabeled iodoalkyl FAs appears limited by: 1) the rapid appearance of free radioiodine, requiring special correction procedures to differentiate between myocardial and blood pool activity; 2) short elimination half-lives, making them unattractive agents for single photon imaging; and 3) data suggesting that the elimination rate may not reflect ⁇ -oxidation but rather de-iodination and back-diffusion of the tracer across the membrane. Further, protocols and algorithms developed for planar imaging are not applicable to single photon imaging (SPECT), effectively eliminating it as a potential imaging modality for the measurement of metabolic parameters with these radiotracers. Imaging difficulties associated with de-iodination of DTDA and DTXA ultimately resulted in the development of the branched FAs.
• SPECT single photon imaging
• the molecular structure of FAs can be modified to attenuate myocardial metabolism, prolong cardiac retention, and avoid washout effects.
• 15-(p- iodophenyl) pentadecanoic acid (IPPA) was developed as an alternative (Machulla, H. et al (1980) Eur. J. Nucl. Med. 5: 171-173).
• IPPA 15-(p- iodophenyl) pentadecanoic acid
• IPPA has the advantages of rapid myocardial uptake, iodine stabilization, and rapid clearance of metabolites from the body.
• IPPA was a significant improvement over the straight chain FAs, providing excellent image quality, and permitting SPECT image acquisition and quantification with estimates of metabolic rates, the rate of IPPA metabolism and clearance was still relatively fast for SPECT imaging. Thus, an effort was made to develop radiolabeled FA analogs with attenuated oxidative metabolism.
• Methyl branching was introduced to slow myocardial clearance and improve quantitative image accuracy (Livni, E. et al. 1982 J. Nucl. Med.23: 169-75; Elmaleh, D.R. et al. 1981. J Nucl. Med. 22: 994-9; Elmaleh, D.R. et al. 1983 Int. J. Nucl. Med. Biol. 10:181-7; Goodman, M.M. et al. 1984 J. Org. Chem. 49: 2322-5; Livni, E. et al. 1985. Eur. Heart J. 6 (Suppl B): 85-9; Bianco, J.A. et al. Eur. J.
• BMffP is currently the most widely used radiolabeled MFA for cardiac imaging.
• BMIPP has prolonged myocardial retention (30-45 minutes) and undergoes ⁇ -oxidation in the myocyte after the initial ⁇ -oxidation and oxidative decarboxylation, producing ⁇ -hydroxy-BMIPP as an intermediate (Yamamichi, Y. et al, (1995) J. Nucl. Med. 36: 1042-1050). After loss of propionic acid, further degradation proceeds through successive cycles of ⁇ -oxidation to the end product, (p-iodophenyl)-acetic acid.
• the present invention relates to novel radiolabeled FA analogs having a cyclic organic substituent, where a positron or gamma-emitting label is placed at a position on an FA backbone and an organic substituent is substituted at the 2,3; 3,4; 4,5; 5,6 and other sequence positions of an FA backbone.
• These novel FA analogs are designed to enter the tissues of interest by the same long chain FA carrier mechanism as natural FAs, however, functional substituents in the 2,3; 3,4; 4,5; 5,6 and other sequence positions, block the catabolic pathway, thus trapping these analogs in the tissues of interest.
• a radioactively labeled analog of a fatty acid that is taken up by mammalian tissue comprising the formula:
• aryl in the context of this application may comprise, but is not limited to, a 5-, 6- or 7-membered ring.
• cyclic can refer to cyclic alkanes such as cyclopropyl, cyclobutyl, and cyclopentyl, but is not so limited.
• heterocylic can refer to any 3 to 5-membered ring structure that can comprise, for example, nitrogen, sulfur, or oxygen atoms.
• a radioactively labeled analog of a fatty acid that is taken up by mammalian tissue comprising the formula:
• D is CH 2 or CH 2 CH 2
• E is CH 2 or CH 2 CH 2
• m is 0-10
• n is 8-14 and R is a CH 3) aryl or a heterocyclic group, wherein cyclic organic substituent -CDCE- causes said analog to be metabolically trapped in said tissue.
• Another aspect of the present invention provides a radioactively labeled analog of a fatty acid, comprising the formula:
• A can be (CH 2 ) ⁇ , O, or S;
• X isl, 2, 3, and 4; cis and trans isomers R,R and S,S and their racemic forms;
• m is 0-10;
• n isl4-8;
• R is 18 F-phenyl, or 123 I-phenyl, and wherein the cyclic or heterocyclic organic substituent -CH-A-CH- causes said analog to be metabolically trapped in said tissue.
• a radioactively labeled analog of a fatty acid that is taken up by mammalian tissue comprising the formula:
• A is (CH 2 ) y , O, or S; y isl, 2, 3, 4; cis and trans isomers R,R, and S,S and their racemic forms; m is 0-10; n isl4-8; p is 0-6; R is CH 3 ; and X is a radioactive label.
• the present invention also provides a radioactively labeled analog of a fatty acid that is taken up by mammalian tissue, comprising the formula:
• X can be H, 18 F, 123 1, 131 1, 34m Cl, 75 Br, 76 Br, 77 Br, alkyl and heteroalkyls thereof;
• Y can be H, 18 F, 123 1, 131 1, 34m Cl, 75 Br, 76 Br, 77 Br, alkyl and heteroalkyls thereof;
• A is (CH 2 ) Z , O or S;
• Z is 1-4, m is 0-10, n is 8-14, p is 0-6,
• R is CH 3 , aryl or heterocyclic, and wherein the cyclic or heterocyclic organic substituent -CH-A-CH- causes said analog to be metabolically trapped in said tissue.
• Another aspect of the present invention provides a radioactively labeled analog of a fatty acid that is taken up by mammalian tissue, comprising the formula:
• A is (CH 2 ) Z , O or S, y is 1-4, m is 1-10, n is 8-14, p is 0-6, R is CH 3 , aryl or heterocyclic, X can be 18 F, 123 1, 131 1, 34m Cl, 75 Br, 76 Br or 77 Br; and wherein the cyclic or heterocyclic organic substituent -CH-A-CH- causes said analog to be metabolically trapped in said tissue.
• the novel fatty acid has a cyclic organic substituent labeled with 18 F, 123 1, 131 1, 34m Cl, 75 Br, 76 Br and 77 Br.
• the novel fatty acid having a cyclic organic substituent is saturated.
• Another embodiment of the present invention describes the novel fatty acid having a cyclic organic substituent that contains one or more double bonds.
• the organic substituent is a 3-to-5 membered cyclic structure.
• Another aspect of the present invention provides a method of measuring blood flow in a subject, comprising the following steps: a) localizing a detectable amount of a FA composition of the invention to a tissue of interest; b) detecting a signal from said FA composition in a tissue of interest within about 1 minutes and about 5 minutes after administration; c) imaging a tissue of interest and; d) determining the rate of blood flow in a tissue of interest.
• a method for measuring metabolism in a subject comprises the following steps: a) localizing a detectable amount of a FA composition of the invention to a tissue of interest; b) detecting a signal from said FA composition in a tissue of interest within about 30 minutes and about 120 minutes after administration; c) imaging a tissue of interest and; d) determining the rate of metabolism in a tissue of interest.
• Another aspect of the present invention provides a method for retaining a composition comprising a fatty acid analog in a tissue of interest, comprising the steps of: a. localizing a detectable amount of the compositions of the present invention to the tissue; b.
• the present invention further provides a method for retaining a fatty acid composition of the invention in a tissue of interest is provided, comprising the steps of a. localizing a detectable amount of the composition to the tissue; b. retaining the composition, or a metabolic derivative thereof, in the tissue by reducing dehydrogenation of the composition; and c. detecting the composition or the metabolic derivative in the tissue.
• Another aspect provides a method for retaining a fatty acid composition in a tissue of interest, comprising the steps of: a. localizing a detectable amount of the composition to the tissue; b.
• the present invention further provides a method for retaining a fatty acid composition in a tissue of interest, comprising the steps of: a. localizing a detectable amount of the composition to the tissue; b. retaining the composition, or a metabolic derivative thereof, in the tissue by reducing ketoacyl formation of the composition; and c. detecting the composition or the metabolic derivative in the tissue.
• Yet another aspect of the present invention provides a method for retaining a fatty acid composition in a tissue of interest, comprising the steps of: a. localizing a detectable amount of the composition to the tissue; b.
• the tissue of interest is heart tissue. In another embodiment, the tissue of interest is liver tissue.
• the present invention preferably describes tumor tissue as the tissue of interest. i one embodiment, the tissue is diseased.
• the tissue is healthy.
• the tissue can be subjected to exercise-induced stress or pharmacologically induced stress.
• Another aspect of the present invention provides a method of synthesizing a fatty acid composition of the invention, comprising the steps of: a) synthesizing a mono-protected primary alcohol from a starting compound; b) adding a cyclic or heterocyclic organic substituent to the mono- protected primary alcohol to form a cyclic mono-protected primary alcohol; and c) treating the cyclic mono-protected primary alcohol to form the fatty acid analog.
• One embodiment of the present invention describes the starting compound comprising a carbon backbone that is saturated.
• the starting compound comprises a carbon backbone that unsaturated.
• Another embodiment describes the starting compound comprising a terminal phenyl group.
• the starting compound is a cyclic primary alcohol.
• One embodiment of the present invention describes a cyclic alkane as the cyclic organic substituent.
• Another embodiment describes the heterocyclic organic substituent as a 3-5- membered heterocyclic ring structure.
• the method further comprises adding a radioactive label that is bonded to a carbon atom of the analog.
• the present invention further provides a kit for administration of a radioactively labeled analog of a fatty acid, comprising an analog of a fatty acid synthesized according to the methods of the invention, a radioactive isotope, a pharmaceutically acceptable carrier, and optionally instructions for preparing the radioactively labeled analog or use thereof.
• the radioactive isotope is selected from the group consisting of 18 F, 123 1, 131 1, 34m Cl, 75 Br, 76 Br and 77 Br.
• Figure 1 is a schematic overview of the synthesis of [ 18 F]-9-fluoro-3,4- cyclopropyl-heptadecanoic acid.
• Figure 2 depicts the general formula of a saturated fatty acid comprising a substituted radiolabel directly on the fatty acid backbone.
• This figure of a fatty acid variant comprises a generalized structure similar to the compound in Figure 1.
• Figure 3 depicts [ 18 F]-FCPHA activity in the heart of a monkey as a function of time.
• Figure 4 shows heart images of a pig at 2-8 minutes after intravenous inj ection of 18 mCi of [ N]-ammon ⁇ a.
• Figure 5 shows heart images of the same pig in Figure 5, at 2-8 minutes after intravenous injection of 19 mCi [ 18 F]-FCPHA.
• Figure 6 is a comparison of [ 13 N]-ammonia (right) and [ 18 F]FCPHA (left) images obtained from a pig at 2-8 minutes after tracer injection.
• Figure 7 is an image collected by positron emission tomography of the biodistribution of [ 18 F]-FCPHA in the left and right ventricles of a monkey heart.
• Figure 8 shows transverse heart level slices of a monkey 60 minutes after administration of [ 18 F]-FCPHA.
• Figure 9 depicts the general formula of a terminally labeled straight chain fatty acid comprising a substituent designated by 'A' and a phenyl moiety comprising a substituted radiolabel.
• Figure 10 is a schematic overview of the synthesis of endo-[ 18 F] fluoro- or
• FIG. 11 depicts the general formula of a fatty acid comprising an endo- vinyl group.
• This endo-vinyl group can comprise a substituted radiolabel at substituents 'X' or ⁇
• Figure 12 is a schematic overview of the synthesis of exo-[ 18 F] fluoro or [ 123 I] iodo-3,4-cyclopropylhe ⁇ tadecanoic acid.
• Figure 13 depicts the general formula of a fatty acid comprising an exo-vinyl group.
• This exo-vinyl group can comprise a substituted radiolabel at substituents ⁇ X' or 'Y'.
• Figure 14 shows the partial synthesis of a portion of a modified fatty acid comprising a substituent that can be a four- to five-membered ring structure.
• Figure 15 shows the general formula of a modified fatty acid comprising a substituent that can be a four- to five-membered ring structure.
• This fatty acid variant can comprise a combination of features described in FIG.2, 4, 11, and 13.
• novel radiolabeled fatty acid compositions having a cyclic organic substituent can be administered to an animal, including a human, to determine both blood flow to the organ and metabolism by an organ of the body of the animal.
• modified fatty acid can be regarded as a synthetic or naturally occurring fatty acid that has been synthetically modified. Also within the context of this application, the term
• organic substituent refers to organic chemical structures bonded to the fatty acid that is effective in decreasing the in vivo rate of ⁇ -oxidation of the fatty acid in tissues of interest.
• the novel FAs described herein are radiolabeled and are modified with an organic substituent at 2,3; 3,4; 4,5; 5,6 and other sequence positions.
• the term "2,3” refers to the carbon bond between the carbon atoms located at position C2 and C3 (counting from the carboxyl carbon atom).
• the related term “3,4" refers to the carbon atoms at positions C3 and C4.
• the term "2,3” could also be interpreted by the skilled artisan to correspond to the term “beta-gamma” in reference to the carbon atoms, or " ⁇ ".
• the related term "3,4" therefore, could be interpreted to correspond to the term "gamma-delta” in reference to the carbon atoms, or " ⁇ ".
• R CH 3 , aryl, heterocyclic
• the organic substituent maybe saturated or unsaturated.
• the organic substituent may also comprise at least one heteroatom, advantageously N, O or S, most advantageously O or S. Nitrogen can also be used and it is well within the skill of the artisan to determine proper substitution of the nitrogen atom, as well as determining the appropriate biodistribution of the resultant analog.
• the carbon backbone of the fatty acid may also be substituted with at least one heteroatom herein defined.
• A can be a -C ⁇ alkyl, alkenyl or alkynyl, wherein one or more of the C atoms, advantageously 1, 2 or 3 C atoms, are substituted by a heteroatom, advantageously N, O or S, most advantageously O or S. It is well within the scope of one skilled in the art to determine proper placement of the nitrogen atom, in addition to determining the appropriate biodistribution of the resultant analog.
• aryl as herein defined may comprise, but is not limited to, a 5-, 6- or 7- membered ring.
• heteroatom as herein defined may comprise, but is not limited to, 1, 2 or 3 heteroatoms.
• the chain length of the FA also affects the tissue by which it is primarily taken up. Generally, a chain length of 12-20 carbon atoms, inclusive, is optimal for selective uptake by myocardial tissue, while the liver will selectively take up a chain length of 5-11 carbon atoms, inclusive.
• the carbon chain of the analog can be saturated or unsaturated.
• a preferred embodiment of the invention is a FA backbone that is saturated and has a cyclic organic substituent.
• the FA backbone contains one or more vinyl groups, resulting in unsaturation of one or more carbon-carbon bonds.
• the vinyl group or groups are placed on the opposite side of the carboxyl group, or to the left of the substituent designated by the letter 'A' in the general formula shown above.
• the vinyl groups are appended to the FA backbone such that the vinylic substituent is branched from the FA chain. More preferably, 1-6 carbon atoms separate the vinyl group(s) from the substituent designated by 'A'.
• Generalized drawings of these preferred embodiments are provided in Figures 6 and 8.
• Activation of FAs is an energy-dependent step necessary for their transport into and sequestration within tissues of interest.
• Preferred embodiments of the instant invention are related to manipulation of the FA metabolism pathway, also referred to herein as " ⁇ -oxidation”.
• This process begins with acyl-coenzyme A (Co A) synthase, which activates cytosolic free FAs by decarboxylation of the terminal COOH at the outer mitochondrial membrane in the presence of adenosine triphosphate (ATP).
• ATP adenosine triphosphate
• This forms an acyl-adenylate mixed anhydride, which reacts with CoA to form fatty acyl-CoA and AMP (adenosine monophosphate).
• CoA acyl-coenzyme A
• ScoA acyl-coenzyme A
• FAs can be "transported” across the imier mitochondrial membranes and undergo stepwise ⁇ -oxidation. Transport across the inner mitochondrial membrane is also referred to in the context of this application as “metabolic trapping" or "metabolic retention” of FAs.
• Activated fatty acyl-CoA cannot be directly transported across the inner mitochondrial membranes and the acyl chain must be transferred to camitine by an acyl-camitine/camitine transporter. This facilitated diffusion through the inner mitochondrial membrane is the rate- limiting step for the oxidation of FAs.
• esterified FAs are diverted into storage pools as cytosolic triglycerides and membrane phospholipids. It is contemplated that activation of the novel analogs described by the invention and transport into the metabolizing tissue of interest occurs normally, but subsequent steps relating to disassembly of the analog are blocked.
• FAs are metabolized in four steps. 1) Formation of a tr ⁇ n5 , -2,3-double bond occurs through acyl-CoA dehydrogenase from a fatty acyl-CoA precursor to form tr ⁇ n,s- ⁇ 2 -enoyl-CoA. 2) The trans double bond is then hydrated by enoyl-CoA hydratase to form 3-L-hydroxyacyl-CoA, which is subsequently 3) dehydrogenated by 3-L-hydroxylacyl-CoA dehydrogenase to form ⁇ -ketoacyl-CoA.
• the present invention relates to FAs having a cyclic organic substituent that cause attenuation of the ⁇ -oxidation pathway by potentially preventing or blocking the metabolic sequence of one or several ways.
• the intermediate is not a substrate for one of the enzymes required during the ⁇ -oxidation pathway or the intermediate cannot undergo a metabolic hydrogenation, dehydrogenation or hydroxylation step.
• An early trapping step that is related to flow and initial uptake may be advantageous for acquiring blood flow related images.
• Metabolic trapping or retention that occurs after one or several metabolic steps can represent the metabolic integrity of the target tissue.
• "Reducing" the formation of metabolic derivatives of the FAs of the invention can mean total or partial prevention or elimination of metabolic reactions such as, but not limited to, transport and back- diffusion, hydrogenation, dehydrogenation, hydroxylation, ketoacyl formation, and ketoacetyl elimination.
• one aspect of the present invention reduces formation of ⁇ -ketoacyl-CoA (step 3).
• Yet another aspect reduces formation of a tra ⁇ 5- ⁇ 2 -enoyl-CoA derivative (step 1).
• Still another aspect of the invention reduces formation of the products of subsequent ⁇ -oxidation steps, depending on the organic substituents and their placement on the FA backbone.
• the position of the organic substituent on the FA backbone can determine the extent of which the molecule undergoes ⁇ -oxidation and metabolism.
• Modifications of the FA can also cause attenuation of FA metabolism.
• a cis-3,4-double bond is not a substrate for enoyl-CoA hydratase and requires enoyl-CoA isomerase, which mediates conversion of the cis double bond to the more stable, ester-conjugated trans form.
• This molecule is a poor substrate for enoyl-CoA hydratase and requires another enzyme, NADPH-dependent 2,4-dienoyl-CoA reductase.
• the resultant molecule, trans-2- enoyl-CoA is further isomerized to yield tr ⁇ .s-3-enoyl-CoA by 3,2-enoyl-CoA isomerase.
• addition of other organic substituents, such as branched methyl or phenyl groups take advantage of ⁇ -oxidation pathway attenuation, which can result in longer retention times in the metabolizing tissue of interest.
• An embodiment of the present invention is substitution of an organic substituent or substituents on the FA chain backbone.
• This organic substituent can be placed at 2,3; 3,4; 4,5; 5,6 and other sequence positions, yielding the addition of the substituent at positions that are branched from the FA backbone.
• the position of the organic substituent on the FA backbone may determine the extent of which the molecule undergoes ⁇ -oxidation and metabolism.
• an organic substituent bonded at the 3,4 position causes the analog to be metabolically trapped in tissues of interest by substitution of CoA for the carboxyl carbon atom of the FA analog.
• the first metabolic step which involves dehydrogenation of the analog to ostensibly form a trans- - enoyl-CoA derivative, does not occur, thus preventing any further metabolism of the analog. It is contemplated that these specific embodiments can be used advantageously to measure blood flow.
• an organic substituent is in position 4,5 and in other subsequent positions on the FA backbone (i.e. 5,6; 6,7; etc), one or more steps in ⁇ -oxidation may be completed and then be subsequently blocked.
• Yet another preferred embodiment of the mvention contemplates appending an organic substituent at positions further on the FA backbone from the carboxyl-terminal end, such as but not limited to the 4,5; 5,6; 6,7 positions and so on. It will be apparent to those skilled in the art that the position of the organic substituent on the FA backbone will determine the extent of which the molecule undergoes ⁇ -oxidation. In this embodiment, metabolic activity of the instant invention can be advantageously measured due to its progression through the ⁇ -oxidation pathway.
• Any organic substituent of the analog should be small enough to permit the formation of the first chemical intermediate involved in the fatty acid ⁇ -oxidation process; too large a substituent can alter the uptake and behavior of an analog to an undesirable extent.
• the chemical nature, as well as the size, of any substituent can affect the properties of the analog.
• an analog having a substituent which does not render the analog excessively polar e.g., an unsubstituted alkyl group
• an analog containing a polarizing group e.g., an alcohol or an ether
• the functional group designated by 'A' in the general formula provided above can comprise branched alkyl groups, dimethyl groups, cyclic alkanes such as cyclopropyl, cyclobutyl, cyclopentyl, or any 3 to 5-membered heterocyclic ring structure.
• the heterocyclic rings can contain nitrogen, sulfur or oxygen atoms.
• a preferred embodiment of the instant invention is a cyclopropyl ring substituent.
• Other preferred embodiments of the invention contemplate cyclobutyl and cyclopentyl ring substituents, such as those described in the general diagram of Figure 10.
• the organic substituent may be appended onto the FA backbone in either cis or trans form.
• a method of cyclopropanation (Simmons-Smith reaction) is described in the Examples section of this application and the improved procedure originates from Charette et al, J Org. Chem. (1995) 60: 1081.
• the components of the reaction can be present as cis or trans isomer, depending on the FA precursor used for the addition of the cyclopropyl moiety.
• the cis diastereomer is preferentially synthesized by using 3 equivalents of the pre-formed complex of Zn(CH 2 I) 2 *DME in CH 2 C1 2 (see Example 1 for details on this complex) and incubation for 3 hours at below -10°C, whereas the cis isomer is made by using the cis olefin and the pre-formed complex and incubating the mixture for 2 hours at below -10°C.
• the cyclopropyl group on the FA backbone has two chiral centers and thus the molecule exists in two enantiomeric forms: R, R or S, S.
• An embodiment of the present invention encompasses each diastereomer and their respective enantiomers, as well as any racemic mixtures or meso compounds thereof.
• the novel fatty acids according to the present invention can be radiolabeled with a positron or gamma-emitting label that is well known in the art.
• Preferred embodiments of the invention comprise 18 F, 123 1, 131 1, 34m Cl, 75 Br, 76 Br or 77 Br.
• radionuclides display different pharmacokinetic properties, such as elimination, clearance from and/or accumulation in biological tissues, and half-life (t ).
• Radionuclides are typically synthesized by a cyclotron, which accelerates subatomic particles from an ion source along a circular orbit. Particle acceleration inside a chamber is controlled by two alternating electromagnetic fields. These accelerated particles can gain energy and collide with a target at close to the speed of light. Bombardment of particles against the target result in unstable, radioactive isotopes, which are then attached to biologically relevant molecules such as those exemplified by the instant invention. Alternatively, commercially available radionuclides are widely used and may be appended onto biologically relevant molecules by chemical synthesis techniques well known in the art.
• radiotracer detection apparatuses such as PET and SPECT scanners, or gamma cameras.
• radiotracer detection apparatuses such as PET and SPECT scanners, or gamma cameras.
• a prefened embodiment of the present invention is addition of a positron or gamma-emitting radiolabel at a position on the FA backbone that prevents significant loss of the radiolabel during FA metabolism and migration to other tissues.
• radiolabeling at the terminal carboxyl group is not recommended, since this carboxyl group is removed by specific enzymes during the early metabolic stages of the FA into the tissues of interest.
• the radiolabel is added to the 9-carbon position of the FA backbone.
• the radiolabel is added to a terminal phenyl group on the FA backbone located on the opposing side from the carboxylic acid group.
• one or more carbon-carbon bonds on the FA backbone is unsaturated, resulting in a vinyl group and the radiolabel is appended directly to the vinyl group.
• a radiolabeled vinyl group is appended onto the FA backbone and is branched from the FA backbone.
• the radiolabeled FAs having a cyclic organic substituent can be synthesized by traditional organic chemical syntheses well known in the art.
• the instant invention may be purified and analyzed by a variety of methods, including column purification, thin-layer chromatography (TLC), reverse-phase chromatography, high-performance liquid chromatography (HPLC), gas chromatography (GC), infrared spectroscopy (DR.), nuclear magnetic resonance (NMR) including variations such as correlation spectroscopy (COSY), nuclear Overhauser effect spectroscopy (NOESY), and rotating frame nuclear Overhauser effect spectroscopy (ROESY), and Fourier Transform, other analytical techniques such as mass spectrometry (MS) and variations thereof, including electrospray, chemical ionization, matrix assisted laser desorption ionization, time-of-flight, fast atom bombardment/liquid secondary ionization, among many other techniques.
• MS mass spectrometry
• compositions comprising the present invention, novel radiolabeled FAs that have cyclic organic substituents, may be in a variety of conventional depot forms.
• conventional depot forms include, for example, solid, semi-solid and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspensions, liposomes, capsules, suppositories, injectable and infusible solutions.
• dosage forms may include pharmaceutically acceptable carriers and adjuvants, which are well known to those skilled in the art.
• These carriers and adjuvants include, for example, RD3I, IS COM, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pynolidone, cellulose-based substances, and polyethylene glycol.
• buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvin
• Adjuvants for topical or gel base forms may be selected from the group consisting of sodium carboxymethylcellulose, polyacrylates, polyoxyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wood wax alcohols.
• the prefened form of the instant invention is an injectable form. Thus, this form may be subject to other treatments during preparation to improve their tolerance in subj ects.
• the animal of interest preferably a human can be injected with the radiolabeled FAs having a cyclic organic substituent.
• Any pharmaceutically acceptable dosage route including parenteral, intravenous, intramuscular, intralesional or subcutaneous injection, maybe used to administer the novel FA compositions of the instant invention.
• the composition may be administered to the subject in any pharmaceutically acceptable dosage form including those which may be administered to a patient intravenously as bolus or by continued infusion over a period of hours, days, weeks, or months, intramuscularly - including paravertebrally and periarticularly — subcutaneously, intracutaneously, intra-articularly, intrasynovially, intrathecally, intralesionally, periostally, or by oral or topical routes.
• the compositions of the invention are in the form of a unit dose and will usually be administered intravenously by bolus injection.
• the purified FA can be contained in solutions such as saline, at concentrations suitable for intravenous delivery. These FAs can be complexed with albumin, a serum protein that binds to non-esterified FAs in the bloodstream.
• albumin is added at a concentration of 2-10%, more preferably between 4- 6%.
• these FAs can be emulsified in non-ionic detergents such as but not limited to, polyoxyethylene sorbitan monolaurate derivatives (Tween), Nonidet P-40, ⁇ -D-octylglucoside, ursodeoxycholic acid (UDCA), or Triton X-100, and resuspended in solutions containing or lacking albumin prior to injection.
• the dosages of radiolabeled cyclic substituted FA must be detemiined for each subject prior to administration, and the typical dosage ranges can be between 0.1-25 mCi, more preferably between 1-5 mCi.
• compositions comprising the FAs according to the present invention may also be administered to any animal, including, but not limited to, horses, cattle, monkeys, birds, pet animals, such as dogs, cats, birds, fenets, hamsters, rodents, squnrels, birds, and rabbits.
• the prefened embodiment of the invention is to monitor diseases or disease states associated with blood flow, FA metabolism or for imaging an organ of interest in a human.
• the present invention further comprises methods to measure and/or identify changes in blood flow and metabolism in tissues of interest in response to disease states, exercise, pharmacological agents, or for diagnostic imaging.
• a method of measuring blood flow in a subject comprises the following steps: a) localizing a detectable amount of the FA composition of the invention to a tissue of interest; b) detecting a signal from said radiolabeled FA composition in a tissue of interest within about 1 minute and about 5 minutes after administration; c) imaging a tissue of interest and; d) determining the rate of blood flow in a tissue of interest.
• the present invention further provides a method of measuring metabolism in a subject comprising the following steps: a) localizing a detectable amount of the FA composition of the invention to a tissue of interest and; b) detecting a signal from said radiolabeled FA composition in a tissue of interest within about 30 minutes and about 120 minutes after administration; c) imaging a tissue of interest and; d) determining the rate of metabolism in a tissue of interest.
• Another embodiment of the present invention provides a method for retaining a fatty acid composition in a tissue of interest, comprising the steps of: a) localizing a detectable amount of the composition to the tissue; b) retaining the composition, or a metabolic derivative thereof, in the tissue by reducing transport and back-diffusion of the composition; and c) detecting the composition or the metabolic derivative in the tissue.
• a method for retaining a fatty acid composition in a tissue of interest comprising the steps of: a) localizing a detectable amount of the composition to the tissue; b) retaining the composition, or a metabolic derivative thereof, in the tissue by reducing dehydrogenation of the composition; and c) detecting the composition or the metabolic derivative in the tissue.
• the present invention further provides a method for retaining a fatty acid composition in a tissue of interest, comprising the steps of: a) localizing a detectable amount of the composition to the tissue; b) retaining the composition, or a metabolic derivative thereof, in the tissue by reducing hydroxylation of the composition; and c) detecting the composition or the metabolic derivative in the tissue.
• Another embodiment of the present invention provides a method for retaining a fatty acid composition in a tissue of interest, comprising the steps of: a) localizing a detectable amount of the composition to the tissue; b) retaining the composition, or a metabolic derivative thereof, in the tissue by reducing ketoacyl formation of the composition; and c) detecting the composition or the metabolic derivative in the tissue.
• Yet another embodiment of the present invention provides a method for retaining a fatty acid composition in a tissue of interest, comprising the steps of: a) localizing a detectable amount of the composition to the tissue; b) retaining the composition, or a metabolic derivative thereof, in the tissue by reducing ketoacetyl elimination of the composition; and c) detecting the composition or the metabolic derivative in the tissue.
• a) localizing a detectable amount of the composition to the tissue comprising the steps of: a) localizing a detectable amount of the composition to the tissue; b) retaining the composition, or a metabolic derivative thereof, in the tissue by reducing ketoacetyl elimination of the composition; and c) detecting the composition or the metabolic derivative in the tissue.
• Numerous methods by which one may use the present mvention in imaging, measurements of blood flow, and FA metabolism are contemplated, used singularly, or in combination with other imaging modalities.
• cardiac imaging modalities generally measure two parameters: blood flow (perfusion) and myocardial vi
• the instant invention can be used in a variety of methods to answer a specific diagnostic question.
• methods can include administration of the invention under conditions of rest and/or stress induced by exercise, disease states, or pharmacological agents.
• the radiolabeled FAs having a cyclic organic substituent can also be used in sequential imaging experiments, depending on the radioisotope used. Blood flow to an organ of an animal can be determined within 1 second to about 10 minutes, preferably between about 1 minute and about 5 minutes after the radiolabeled cyclic substituted FA is administered to the animal. Metabolism by the organ of interest can be determined within a time period between about 10 minutes to about 24 hours, preferably between about 30 minutes and about 120 minutes after administration of the radiolabeled FA into the bloodstream of the animal.
• Nuclear cardiac imaging using the present invention can be used to detect a wide variety of cardiac derangements.
• cyclic substituted, radiolabeled FAs can be used singularly as a marker for blood flow and for cardiac metabolism, or may be used in combination with another tracer.
• Such tracers include, but are not limited to N- Ammonia, Co- Cyanocobalamin, 5 Fe-Fenous Citrate, 18 F-Fluorodeoxyglucose, 67 Ga-Gallium Citrate, 11 ⁇ -Indium Oxyquinoline, 11 ⁇ -Indium Pentetate, l x ⁇ -Indium
• the tissues of interest can be any tissues that utilize FAs as a source of energy.
• the tissues can be, but are not limited to, cardiac tissue, brain, liver, bone, spleen, lung, blood, kidney, gastrointestinal, muscle, and adrenal tissue.
• a prefened embodiment of the present invention is cardiac tissue.
• Another prefened embodiment can also encompass liver tissue. Cardiac diseases or disease states that can be monitored using radiolabeled
• FAs having cyclic organic substituents include, but are not limited to, acute myocardial infarction, unstable angina, chronic ischemic heart disease, coronary artery disease, myocarditis, dilated, hypertrophic, and restrictive cardiomyopathies, congenital heart diseases, hypertensive heart disease, post-transplant heart disease, allograft vasculopathies, valvular heart disease, and pharmacologically induced conditions such as doxorubicin cardiotoxicity. It is contemplated that methods of use of radiolabeled FAs exemplified by the present invention will be modified according to the particular disease examined.
• the instant invention can also be used in numerous non-cardiac disease states described herein, such as: abscess and infection; biliary tract blockage; blood volume studies; blood vessel diseases; blood vessel diseases of the brain; bone diseases; bone marrow diseases; brain diseases and tumors; cancer and neoplasms; colorectal disease; diabetes; disorders of iron metabolism and absorption; heart disease; heart muscle damage such as infarction and ischemia; impaired flow of cerebrospinal fluid in brain; kidney diseases; liver diseases; lung diseases; parathyroid diseases and/or parathyroid cancer; pernicious anemia and/or improper absorption of vitamin B 12 from intestines; red blood cell diseases; salivary gland diseases; spleen diseases; stomach disorders and intestinal bleeding; tear duct blockage, thyroid diseases and/or thyroid cancer, urinary bladder diseases.
• non-cardiac disease states described herein such as: abscess and infection; biliary tract blockage; blood volume studies; blood vessel diseases; blood vessel diseases of the brain; bone diseases; bone
• a prefened embodiment of the present invention is its use in detecting cardiac myopathies by measuring blood flow and FA metabolism. It is understood that diagnosis of the aforementioned diseases will often require the use of other radiotracers, also described above. It is apparent that use of the instant invention is not only limited to detection of diseased states, but also for diagnostic imaging in healthy subjects.
• Positron Emission Tomography PET
• SPECT Single Photon Emission Computed Tomography
• PET and SPECT rely on similar principles to produce their images, important differences in instrumentation, radiochemistry, and experimental applications are dictated by inherent differences in their respective physics of photon emission.
• Unstable nuclides that possess an excess number of protons may take one of two approaches in an effort to reduce their net nuclear positivity.
• a proton is converted to a neutron and a particle called a positron is emitted
• positrons are the antimatter equivalent of electrons.
• a positron collides with an electron, resulting in the annihilation of both particles and the release of energy.
• Two ⁇ photons are produced, each of equivalent energy and opposite trajectory (generally 180° apart).
• PET and
• SPECT scanners employ scintillation detectors made of dense crystalline materials (e.g., bismuth germanium oxide, sodium iodide, or cesium fluoride), that capture the high-energy photons and convert them to visible light. This brief flash of light is converted into an electrical pulse by an adjacent photomultiplier tube (PMT). The crystal and PMT together make up a radiation detector.
• a PET camera is constructed such that opposing detectors are electronically connected. Thus, when separate scintillation events in paired detectors coincide, an annihilation event is presumed to have occuned at some point along an imaginary line between the two. This information is used to reconstruct images using the principles of computed tomography. Conversely, single events are ignored.
• PET recognizes the site of positron annihilation, which does not necessarily coincide with the site of radioactive decay. Annihilation often occurs some distance away from the positron's origin. The distance separating these two events, decay and annihilation, depends on the average kinetic energy of the positron as it leaves the nucleus, and varies according to the specific isotope involved (Phelps ME,et al. J Nucl Med (1975); 16: 649-652). In addition, if the positron is not entirely at rest at annihilation, photons will be emitted at an angle slightly different than 180°. Taken together, remote positron annihilation and photon non-colinearity place a theoretical limit on PET's achievable spatial resolution (Links JM. New York: Raven Press; (1990): 37-50).
• isotopes that decay by electron capture and/or ⁇ emissions can be directly detected by SPECT.
• Certain proton-rich radionuclides such as 123 I and 99m Tc, may instead capture an orbiting electron, once again transforming a proton to a neutron (Sorenson JA, and Phelps ME. Philadelphia: W.B. Saunders; 1987).
• the resulting daughter nucleus often remains residually excited.
• This meta-stable anangement subsequently dissipates, thereby achieving a ground state and producing a single ⁇ photon in the process. Because ⁇ photons are emitted directly from the site of decay, no comparable theoretical limit on spatial resolution exists for SPECT.
• SPECT utilizes a technique known as colhmation (Jaszczak RJ. Boca Raton: CRC Press; (1991): 93-118).
• a collimator may be thought of as a lead block containing many tiny holes that is interposed between the subject and the radiation detector. Given knowledge of the orientation of a collimator's holes, the original path of a detected photon is linearly extrapolated and the image is reconstructed by computer-assisted tomography.
• the assumption is such that at steady state, the rate at which FA leaves the tissue through oxidation must be equal to the rate at which FA is entering the tissue from the blood.
• the net extraction of a substance metabolized by the heart in a steady state can be determined from the arterial or arterialized blood and venous blood concentrations of FAs.
• L here plays the same role as the "lumped constant" in Sokoloff s model for deoxyglucose metabolism (Sokoloff, L. et al (1977) J. Neurochem. 28: 879-916). The difference between the two models is that L may not be constant. 'L' can vary with blood flow, free fatty acid concentration, FFA composition, or other physiological parameters. The behavior of L is best understood by performing a series of experiments to determine the relationship between E n and E n [FA analog] in different physiological states.
• Measurements with FA analogs can only determine the rate at which FA analog passes through the committed step to acyl-CoA. Accordingly, the steady state rate for incorporation into triglyceride plus ⁇ -oxidation can be measured. This situation is analogous to the result that would be obtained if Fick-type atrial/ventricular measurements with a native fatty acid such as palmitate could be made.
• an operational equation capable of describing the PET measurements in terms of the blood flow is described, F being the unidirectional extraction fraction, E as the net extraction fraction, E n , and the rate K, with which FA analog is cleared from the "precursor pool".
• the rate constant K is the sum of two rates, K 2 the rate of back-diffusion, and K 3 , the rate of activation of FA analog to the CoA form.
• C(t) is the tissue concentration at time t
• Ca(t) is the plasma concentration at time t
• (*) represents the mathematical operation of convolution.
• E n E*K3/K2+K3.
• E is the probability a FA analog molecule will leave the blood on a single capillary transit
• K3/(K2 + K3) is the probability that a FA analog molecule entering the tissue will be metabolically trapped.
• the resulting compound, l-trityl-6-tetradecanol (20 g, 42.3 mmol), was slowly mixed with 60% sodium hydride (2 g, 50 mmol) and benzyl bromide (7.2 g, 42.3 mmol) in dry dimethylformamide (DMF; 100 ml) in a water bath set at RT.
• the mixture was stined for 4 hours at RT and poured into ice water, then extracted with ether. Chromatography of the crude oil on silica gel using hexane:ethyl acetate (90:10) yielded 19 g (34 mmol), or 80% of the diether compound, 6-benzyloxy-l- trityl-tetradecane diether.
• This diether compound was mixed with 100 mg ofp- toluene sulfonic acid in methanol (100 L) at RT for 4 hours.
• Sodium bicarbonate 100 mg was added and the solvent removed by rotary evaporation.
• the oil was separated by chromatography on silica gel in hexane:ethyl acetate (80:20) yielded 7.5 g, or 90% of the alcohol.
• PCC (9.7 g, 45 mmol) was added in portions to a solution containing 6- benzyloxy-1-tetradecanol (7.5 g, 30 mmol) in methylene chloride (100 ml) at RT.
• the black mixture was stined for 2 hours, then filtered through a bed of silica gel using he ⁇ ane:ethyl acetate (90:10).
• the conesponding aldehyde, 6-benzyloxy- tetradecanal was produced at a yield of 6.1 g (81%).
• This aldehyde compound was mixed with carbethoxymethylene triphenylphosphorane (10 g, 29 mmol) in 100 ml of methylene chloride at RT.
• DD3AL diisobutylaluminum hydride; 8 ml, 44 mmol
• ester compound 8-benzyloxyhexadec-2-enoate ethyl ester 8.4 g, 22 mmol
• the mixture was allowed to reach 0°C and 5 ml of ethyl acetate was added dropwise, followed by ice.
• the resultant slurry was acidified with 10% HCl, followed by ether extraction. The combined ether extracts were washed with brine, dried, and the solvent was removed.
• Diiodomethane (6 ml) was added dropwise to the solution, while maintaining the reaction temperature between -25°C and -10°C. This solution was then added by double-ended needle, to a solution containing 8-benzyloxyhexadec-2-enol (2 g, 5.8 mmol), dioxaborolane (made from (+)N N N', N'-tetramethyl-L-tartaramide and butylboronic acid), and 300 mg of 4 A molecular sieves in methylene chloride (40 ml) under nitrogen at - 40°C and -30°C. The reaction mixture was stined for 2 hours at -25°C, then allowed to warm to 0°C.
• DMSO dimethyl sulfoxide
• the radiolabel F contained in water (60 mCi, 1 ml), was added to a vial containing 10 mg of Kryptofix-222 (4,7, 13 , 16,21 ,24-hexaoxa- 1,10- diazabicyclo[8.8.8]hexacosane; Merck, Whitehouse Station, NJ) and 4 mg of K 2 CO 3 . Water was removed using a stream of nitrogen at 115°C, folowed by the three rounds of addition of acetonitrile (2 ml).
• the mesylate ester, 9-hydroxy-3,4- cyclopropylheptadecanoate methyl ester (10 mg) in 1 ml of acetonitrile was combined with the radiolabel mixture and heated for 10 minutes in 120°C. This yielded the 18 F-labeled ester at 80% yield after silica Sep-Pak purification (Waters Corporation, Milford, MA) in hexane:ethyl acetate (85:15).
• the labeled ester was placed in a vial and the solvent was removed. It was replaced with 0.1 ml of 1M lithium hydroxide (LiOH) and 0.3 ml of methanol. The reaction vial was heated to 60°C for 20 minutes.
• a schematic representation of the organic synthesis described above is shown in Figure 1.
• the labeled fatty acids were formulated in 10% ethanol in saline for rat studies and 4% BSA in saline for monkey studies (sterile filtered through a 0.22 ⁇ m Millipore filter). i n 1 O
• Example 2 Biodistribution of F-labeled 3,4-cyclopropyl-heptadecanoic acid( F]- FCPHA)
• the initial myocardial behavior of [ 18 F]-FCPHA was essentially similar to that of normal FAs, as evidenced that this analog concentrates in the same metabolic pools and has the same subcellular distribution in the rat heart, where it is found in the mitochondria.
• the radiolabeled FA was injected into the tail vein and the rats subsequently sacrificed at 5 minutes and 60 minutes post-injection.
• the organs were excised and counted in a gamma counter. Accumulation of the FA in various organs in %DPG (dose per gram) is detailed in Table 1.
• Table 3 shows the biodistribution of the ⁇ -methyl analog, [ 18 F]FBMHA, at 5 and 60 minutes after intravenous administration in rats (5 per time point).
• accumulation of radioactivity in the heart was 2.56% dose per gram, with nearly an equal amount of radioactivity in the kidneys.
• Tracer activity in the blood at 5 minutes was 1.02% DPG, and remained relatively high (0.58% DPG) after 60 minutes. Most of the radioactivity accumulated in the liver.
• the heart-to- blood ratio of 2.6 at 5 minutes did not significantly change after 60 minutes.
• Biodistribution of [ 18 F]FBMHA in rats was repeated using a 4% Tween-80/saline formulation and similar results were obtained.
• Table 4 represents heart-to-blood ratios in rats 60 minutes after administration of labeled [ 18 F]FCPHA compared to other ⁇ -methyl analogs [ 18 F]FBMHA, [ ⁇ C]BMHA, and [ 125 I]BMffP.
• Figure 3 represents heart images of a pig at 2-8 minutes post- injection of [ 13 N]ammonia (18 mCi), and Figure 5 displays heart images of the same
• the brominated compound in ether was injected into refluxed ether containing magnesium. Reflux continued for 90 minutes under an argon or nitrogen atmosphere. The reaction mixture was then cooled to room temperature, then subsequently injected into a 7 CO 2 trap and the solution shaken for 5 minutes. The solution was transfened to a separatory funnel, washed twice with ether, and combined with IN HCl. The solution was washed twice with water, dried using Na 2 SO 4 and evaporated. The resulting compound was predicted to be 17-phenyl-l- heptadecanol. The following steps after the second PCC oxidation will continue as described in Example 1. This synthesis yields an analog that contains a terminal phenyl group and a cyclopropyl moiety at the 3,4-carbon position (see FIG.9).
• the 18 F or 123 I radiolabel can be added to the terminal phenyl moiety by other methods, such as the Schiemann reaction.
• the benzene moiety can undergo nitration in nitric acid (HNO 3 ) and sulfuric acid (H 2 SO 4 ), followed by reduction with Sn and HCl. This yields a phenyl moiety labeled with an amino group (aniline).
• Incubation with NaNO 2 and HCl will convert the amino group to ⁇ o the diazonium ion.
• the diazonium salt is then subjected to fluonnation with [ F]- HBF 4 . This yields a phenyl group mono-substituted with fluorine.
• the benzene substituent will undergo nitration in HNO 3 and H 2 SO 4 , yielding an aniline group.
• NaNO 2 and HCl followed by iodination with potassium iodide (KI).
• KI potassium iodide
• the iodinated aryl derivative is radiolabeled by an exchange reaction with radioiodide in an acid media at high temperatures.
• Other methods include preparation of a conesponding tributyl-tin derivative, followed by electrophilic aromatic radioiodination.
• the unsaturated primary alcohol was subjected to cyclopropanation essentially as described in Charette and coworkers, as well as Example 1. Dry methylene chloride was cooled to -25°C and placed under a nitrogen atmosphere. Diethyl zinc, followed by 1,2-dim ⁇ thoxyethane (DME), was added. Diiodomethane was added dropwise to the solution, while maintaining the reaction temperature between -25°C and -10°C.
• DME 1,2-dim ⁇ thoxyethane
• the conesponding nitrile compound was mixed with KOH and water in ethylene glycol, then heated for 6 h at 170°C. Once cooled, the mixture was diluted with 10% HCl and extracted with ether. The combined extract was dried and the solvent removed.
• the crude carboxylic acid was treated with diazomethane (made from N-methyl-N'-nitro-N-nitrosoguanidine and 40% KOH in ether). The reaction mixture was stined for 1 h before solvent removal. Chromatography of the crade oil on silica gel and hexane/ethyl acetate (80:20) yielded the conesponding methyl ester.
• the methyl ester was hydrogenated using lithium aluminum hydride, once again yielding a primary alcohol.
• the primary alcohol was substituted by addition of an alcohol-protecting group, tetrahydropyran (THP).
• THP tetrahydropyran
• the alkyl bromide variant was then subjected to treatment with n- butyllithium in hexane, in the presence of an alkyne, which appended the alkyne group opposite from the THP moiety.
• the conesponding cyclopropyl alkyne was hydrogenated with tributyl-tin hydride and iodinated with I 2 to yield an endo-vinyl variant.
• the endo-vinyl variant molecule was treated with TosH and CrO 3 under acidic conditions to facilitate removal of the THP protecting group and subsequent oxidation to the carboxylic acid.
• the conesponding carboxylic acid was then subjected to substitution of the iodide with tributyl-tin hydride.
• Radiolabeled sodium iodide (Na I) afforded substitution of the tributyl-tin moiety with I, resulting in endo-[ 123 I]-iodo-3,4-cyclopropylheptadecanoic acid.
• the addition of the radiolabeled iodine can be directly achieved, thereby eliminating the intervening vinylic tributyl-tin precursor molecule ( Figure 12).
• the molecule can then be reacted with copper and Na 123 I directly, resulting in exo-[ 123 I]- iodo-3 ,4-cyclopropyl-heptadecanoic acid.
• the vinylic tributyl-tin fatty acid derivative described in Scheme 1 of vinylic iodination can also be used to directly fluorinate the modified fatty acid by substitution of 18 F 2 at the vinylic tributyl-tin moiety ( Figure 12). This yields exo- [ 18 F] fluoro-3 ,4-cyclopropyl-heptadecanoic acid.
• a separate flask was equipped with a constant-pressure addition funnel fitted with a nitrogen inlet, and a water-cooled reflux condenser topped with a T joint leading to a mineral oil bubbler.
• the apparatus was flushed with nitrogen, flame- dried and allowed to cool to room temperature while de-aeration with nitrogen was continued.
• the flask was charged with fluoromethyltriphenylphosphonium iodide, dry THF, and toluene.
• the nitrogen atmosphere was maintained throughout the system.
• the slurry of phosphonium salt and solvent was moderately stined while cooled in a dry ice-isopropanol slush bath.
• the addition funnel was charged with n-butyllithium in hexane.
• the conesponding ketone obtained from PDC oxidation of the methyl ester 9-hydroxy-3,4-cyclopropylheptadecanoate was then added .
• the reaction between the ylide and the ketone proceeded for 2 h at -78°C, then allowed to warm to room temperature, where it was incubated for an additional 1.5 h.
• the reaction mixture was then cooled on ice. Potassium tert-butoxide was added and the mixture was stined for 2 h at 0°C.
• the reaction mixture was centrifuged and decanted, and the precipitate washed with small portions of THF.
• the cyclic mono-protected nonanol was subjected to PCC oxidation, yielding the conesponding aldehyde. Under a nitrogen atmosphere, octyl bromide in dry ether was added to magnesium metal in ether at a rate as to maintain gentle reflux. After addition was complete, the reaction mixture was stined for 1 h and the cyclic mono-protected nonanal in ether, was added dropwise to the reaction mixture at room temperature. The mixture was stined for 4 h, then poured over ice water, acidified in 10% HCl, and extracted with ether. The combined extracts were washed wth brine, dried, and solvent removed.
• the mixture was stined for 2 h, then washed with 10% HCl until the aqueous layer was acidic and then washed with 10% NaHCO 3 .
• the crade oil was chromatographed on silica gel in methylene chloride/methanol (95:5). 18 Fluorine in water was added to a vial containing Kryptofix-222 and K 2 CO 3 . Water was removed using a nitrogen stream at 115°C followed by addition of acetonitrile. To this vial was the mesylated carboxylic acid in acetonitrile.
• synthesis of this molecule can be achieved by using a different starting material (such as 10- phenyl-l-decanol).
• This starting material, 10-phenyl-l-decanol was reacted with triphenylphosphine and dissolved in benzene.
• a solution of carbon tetrabromide in benzene was added slowly and the mixture was refluxed for 90 minutes.
• the reaction mixture was then cooled and filtered, and the residue washed three times with portions of petroleum ether. The residue was evaporated to dryness and then stined with petroleum ether and left overnight in a freezer.
• the brominated compound was subjected to Grignard synthesis as follows.
• the brominated compound in ether was injected into refluxed ether containing magnesium. Reflux continued for 90 minutes under an argon or nitrogen atmosphere.
• the reaction mixture was then cooled to room temperature, then subsequently injected into a trap containing a cyclobutyl- or cyclopentyl mono- protected 1 -heptanol and the solution shaken for 5 minutes.
• the solution was transfened to a separatory funnel, washed twice with ether, and combined with IN HCl.
• the solution was washed twice with water, dried using Na 2 SO 4 and evaporated.
• the resulting heptadecanol compound was predicted to contain a terminal phenyl group at one end, with a cyclobutyl or cyclopentyl substituent followed by the THP protective group.
• the cyclic mono-protected heptadecanol was then subjected to de-protection by treatment with TosH and CrO 3 . This yielded the formation of the conesponding cyclic carboxylic acid.
• the cyclic carboxylic acid was combined with methane sulfonyl chloride and 4-dimethyl aminopyridine (DMAP) in methylene chloride/pyridme (90:10). The mixture was stined for 2 h, then washed with 10% HCl until the aqueous layer was acidic and then washed with 10% NaHCO 3 .
• the crude oil was chromatographed on silica gel in methylene chloride/methanol (95:5).
• the 18 F or 123 I radiolabel can be added to the terminal phenyl moiety by other methods, such as the Schiemann reaction.
• the benzene moiety can undergo nitration in nitric acid (HNO 3 ) and sulfuric acid (H 2 SO 4 ), followed by reduction with Sn and HCl. This yields a phenyl moiety labeled with an amino group (aniline).
• Incubation with NaNO 2 and HCl will convert the amino group to the diazonium ion.
• the diazonium salt is then subjected to fluorination with [ 18 F]- HBF 4 . This yields a phenyl group mono-substituted with fluorine.
• the benzene substituent will undergo nitration in HNO 3 and H 2 SO 4 , yielding an aniline group.
• NaNO 2 and HCl followed by iodination with potassium iodide (KI).
• KI potassium iodide
• the iodinated aryl derivative is radiolabeled by an exchange reaction with radioiodide in an acid media at high temperatures.
• Other methods include preparation of a conesponding tributyl-tin derivative, followed by electrophilic aromatic radioiodination.
• Cyclobutyl and cyclopentyl variants of the endo-vinylic modified fatty acids described in Example 5 are synthesized essentially as described, however the starting material, a cyclobutyl- or cyclopentyl THP mono-protected heptanol, was treated with N-bromosuccinimide. This yielded an alkyl bromide variant, wherein the bromine group was appended on the opposite end from the THP protecting group. The alkyl bromide variant was then subjected to treatment with n- butyllithium in hexane, in the presence of an alkyne, which appended the alkyne group opposite from the THP moiety. The conesponding cyclopropyl alkyne was hydrogenated with tributyl-tin hydride and iodinated with I 2 to yield an endo-vinyl variant.
• the endo-vinyl variant molecule was treated with TosH and CrO 3 under acidic conditions to facilitate removal of the THP protecting group and subsequent oxidation to the carboxylic acid.
• the conesponding carboxylic acid was then subjected to substitution of the iodide with tributyl-tin hydride.
• Radiolabeled sodium iodide (Na 123 I) afforded substitution of the tributyl-tin moiety with 123 I, resulting in endo-[ 123 I]-iodo-3,4-cyclobutyl- or cyclopentyl-heptadecanoic acid.
• the tributyl-tin substituted carboxylic acid was treated with 18 F 2 to yield endo-[ F]-fluoro-3,4-cyclobutyl- or cyclopentyl-heptadecanoic acid.
• the cyclic mono-protected nonanol was subjected to PCC oxidation, yielding the conesponding aldehyde. Under a nitrogen atmosphere, octyl bromide in dry ether was added to magnesium metal in ether at a rate as to maintain gentle reflux. After addition was complete, the reaction mixture was stined for 1 h and the cyclic mono-protected nonanal in ether, was added dropwise to the reaction mixture at room temperature. The mixture was stined for 4 h, then poured over ice water, acidified in 10% HCl, and extracted with ether. The combined extracts were washed wth brine, dried, and solvent removed.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Pharmacology & Pharmacy (AREA)
• Veterinary Medicine (AREA)
• Chemical & Material Sciences (AREA)
• Public Health (AREA)
• General Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Animal Behavior & Ethology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Physics & Mathematics (AREA)
• Epidemiology (AREA)
• Optics & Photonics (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Engineering & Computer Science (AREA)
• Organic Chemistry (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Measuring Pulse, Heart Rate, Blood Pressure Or Blood Flow (AREA)

Priority Applications (7)

Applications Claiming Priority (2)

Publications (2)

Family

ID=33310796

Family Applications (1)

Country Status (9)

Cited By (9)

Families Citing this family (35)

Family Cites Families (12)

• 2004
  • 2004-04-19 MX MXPA05011189A patent/MXPA05011189A/es active IP Right Grant
  • 2004-04-19 EP EP04760019A patent/EP1622602B1/en not_active Expired - Lifetime
  • 2004-04-19 CA CA2522737A patent/CA2522737C/en not_active Expired - Fee Related
  • 2004-04-19 JP JP2006513138A patent/JP4633052B2/ja not_active Expired - Fee Related
  • 2004-04-19 WO PCT/US2004/012084 patent/WO2004093650A2/en not_active Ceased
  • 2004-04-19 AT AT04760019T patent/ATE405258T1/de not_active IP Right Cessation
  • 2004-04-19 DE DE602004015970T patent/DE602004015970D1/de not_active Expired - Lifetime
  • 2004-04-19 US US10/827,054 patent/US7494642B2/en not_active Expired - Fee Related
  • 2004-04-19 AU AU2004232297A patent/AU2004232297B2/en not_active Ceased
• 2007
  • 2007-10-10 US US11/973,810 patent/US20080214853A1/en not_active Abandoned
  • 2007-10-11 US US11/974,139 patent/US7790142B2/en not_active Expired - Lifetime
  • 2007-10-11 US US11/973,926 patent/US20080214851A1/en not_active Abandoned
  • 2007-10-11 US US11/973,921 patent/US20080095702A1/en not_active Abandoned
  • 2007-10-11 US US11/973,922 patent/US8309053B2/en not_active Expired - Fee Related
  • 2007-10-11 US US11/973,925 patent/US20080213178A1/en not_active Abandoned
• 2009
  • 2009-02-23 US US12/391,250 patent/US8268291B2/en not_active Expired - Fee Related
• 2012
  • 2012-08-27 US US13/595,546 patent/US8871179B2/en not_active Expired - Lifetime

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events