Patent Application
20220160905
The invention relates to a process for producing a compound comprising the anion [CF218FSO2]−, and to the compound itself, which comprises that anion. The invention also relates to the use of the compound comprising the anion [CF218FSO2]− to produce a compound comprising an 18F-trifluoromethyl functionalised aromatic group. Compounds comprising an 18F-trifluoromethyl functionalised aromatic group are also the subject of the present invention.
Positron Emission Tomography (PET) is a powerful molecular imaging technique for diagnosis, monitoring disease progression, studying biological processes in vivo and investigating the efficacy of drugs. Among all the radioisotopes required for the preparation of PET probes, 18F is the most widely used and clinically relevant radionuclide. Due to its short half-life (t1/2 109.7 min), 18F must be incorporated into tracer molecules at a late stage of the overall synthesis process. Additional challenges imposed by radiochemistry include low reaction concentrations, solvent compatibility, and cyclotron-produced 18F sources being limited to 18F-fluoride and [18F]F2 only. These constraints are particularly stringent for biomolecules.
Peptides and proteins display excellent binding specificities to targets linked to disease states. Much effort has been made towards developing 18F-labeling methods for these probes (Richter, S., et al., Molecules 2014, 19 (12), 20536-20556). 18F has been incorporated into pre-functionalized peptides and proteins via C-18F, B-18F and Si-18F bond formation, or chelation with Al-18F (see Bernard-Gauthier, V. et al., Biomed Res. Int. 2014, 2014, 454503; Laverman, P. et al. J. Label. Compd. Radiopharm. 2014, 57 (4), 219-223; Cornilleau, T. et al., Org. Lett. 2015, 17 (2), 354-357; Perrin, D. M. Acc. Chem. Res. 2016, 49 (7), 1333-1343). Alternatively, an 18F-labeled prosthetic group is prepared prior to bioconjugation under mild reaction conditions (see Marik, J., et al. Tetrahedron Lett. 2006, 47 (37), 6681-6684; Gao, Z. et al. J. Am. Chem. Soc. 2013, 135 (37), 13612-13615; Jacobson, O. et al., X. Bioconjug. Chem. 2015, 26 (10), 2016-2020; Way, J. D. et al., Chem. Commun. 2015, 51 (18), 3838-3841; Chiotellis, A. et al., T. L. Chem. Commun. 2016, 52 (36), 6083-6086). Attaching the 18F-labeled prosthetic group may be achieved by synthetic manipulation of the biomolecule to enable attachment of the 18F-prosthetic group. Alternatively, the 18F-labeled prosthetic group may be attached to the peptide or protein by taking advantage of the inherent nucleophilicity of amino acid side-chains such as cysteine thiols (see Chalker, J. M. et al., Chem.—An Asian J. 2009, 4 (5), 630-640) or lysine amines. Existing 18F-labeling strategies for unmodified peptides or proteins require the radiosynthesis of 18F-prosthetics followed by a bioconjugation process that harnesses the reactivity of heteroatom lone-pair nucleophiles such as cysteine thiols or lysine amines. This limits the nature of functional groups on which 18F-functionalisation can be performed.
Although tolerated for many applications, these 18F-labeling strategies can significantly alter the structure of native probes. Efficacy and/or function can be adversely affected, for example by changing pharmacokinetic profiles, disrupting hydrogen-bonding interactions or disturbing local polarity. As such, the discovery of 18F-labeling methods targeting native aromatic amino acid residue in peptides or proteins with either 18F, the smallest possible 18F-tag, or a minimally sized 18F-prosthetic (e.g. [18F]CF3) would be of considerable value to many scientists.
A method for modifying cysteine thiols with 5-18F-(trifluoromethyl)dibenzothiophenium trifluoromethanesulfonate to allow 18F-trifluoromethylation of native peptides has been reported by Verhoog et al. (Verhoog et al., 18F-Trifluoromethylation of unmodified peptides with 5-18F-(Trifluoromethyl)dibenzothiophenium Trifluoromethanesulfonate, J. Am. Chem. Soc. 2018, 140, 1572-1575). This method only however permits functionalization of nucleophilic thiol groups, thereby limiting the possible sites of functionalization and the types of molecules that can be functionalised.
In Imiolek et al. (Imiolek, M., Selective Radical Trifluoromethylation of Native Residues in Proteins, J. Am. Chem. Soc 2018, 140, 1568-1571) tuned radical chemistry is applied to program C—H 19F-trifluoromethylation of innately electron rich residues in proteins. Sodium trifluoromethanesulfinate (NaTFMS, Langlois' reagent) displayed selective reactivity for tryptophan under redox initiation. However, as outlined above, a stringent set of considerations apply when preparing and using “hot” 18F reagents as opposed to their 19F counterparts. In particular, methods must be developed to synthesise the reagents quickly and in high yield from cyclotron 18F sources, i.e. 18F−. The reagents synthesised must then be capable of reacting quickly and cleanly with the molecule to be labelled. Due to the short half life of 18F, speed and simplicity of reaction is critical to ensure that 18F can be effectively used to monitor biological processes/systems, for instance through PET.
Ichiishi et al. (Ichiishi, N., Protecting group free radical C—H trifluoromethylation of peptides; Chem. Sci., 2018, 9 (17), 4168-4175) demonstrated that Zn(TFMS)2 (Baran's reagent) when activated with a stoichiometric oxidant or via visible photoredox catalysis, enables trifluoromethylation of tyrosine in peptides that do not contain tryptophan residues. This paper is concerned with standard 19F chemistry only therefore does not address the particular set of problems for developing a “hot” 18F version of the TFMS ion as outlined above.
Routes towards trifluoromethanesulfinic acid salts are known, including metal or electro-reduction of a mixture of SO2 and CF3Br in DMF (see Folest, J.-C et al., Synth. Commun. 1988, 18 (13), 1491-1494), the treatment of CF3Cl with sodium dithionite (Na2S2O4) (see Cao, H. P. et al., J. Fluor. Chem. 2007, 128 (10), 1187-1190), or multistep synthesis featuring a key β-elimination process from trifluoromethylsulfone precursors (see Langlois, B. R. et al., C. J. Fluor. Chem. 2007, 128 (7), 851-856). A schematic of each of these known routes is provided below in scheme 1A.
For radiochemistry, these approaches are unsuitable because they are convoluted. First, they would require a radiosynthetic route towards the necessary [18F]CF3-precursor (for instance CF218FBr or CF218FCl), and then one or more reactions post-labeling would be needed to form the [CF218FSO2][M] product. It is not thought that these routes could successfully be employed to produce [CF218FSO2]− in the first place, but even if any one of them could successfully be used the route would still be too convoluted and time-consuming for radiochemistry. In addition, further steps would be required to functionalise the molecule of interest, giving a minimum of three steps in the overall reaction pathway to the 18F-labelled product. Such a route would be too time consuming and complex given the short half-life of 18F; by the time the desired 18F-labelled product were synthesized positron emission would have decayed to levels unsuitable for use in PET imaging.
The present invention provides access for the first time, to compounds comprising the anion [CF218FSO2]−. This has been achieved by developing a new process that is successfully able to produce 18F-trifluoromethanesulfinate, in a fast and reliable synthesis. The synthesis is performed by combining multiple reactants: a source of 18F-fluoride, a difluorocarbene source, and a source of SO2. The invention therefore addresses the issues discussed above, by providing a quick and facile route to 18F-trifluoromethanesulfinate, which may be performed in a single step. The simple one-pot nature of the process of the invention makes it highly suitable for preparing this “hot” reagent quickly, so that it can then be used to radiolabel molecules of interest, such as peptides and proteins.
The invention therefore provides a fast and reliable route for synthesis of [CF218FSO2]−, to permit facile radiolabelling of aromatic groups. Indeed, the “hot” 18F-trifluoromethanesulfinate reagent permits direct [18F]CF3-incorporation at aromatic groups, such as those that are present in tryptophan and tyrosine residues in unmodified peptides as complex as human insulin. This functionalization process utilises electrophilic radical chemistry to target (hetero)aromatic residues with an 18F-trifluoromethyl group. The ability of 18F-trifluoromethanesulfinate to enable selective C—H 18F-trifluoromethylation of aromatic groups, such as those found on amino acid residues within unmodified peptides and proteins is also demonstrated herein.
Accordingly, the invention provides a process for producing a compound comprising the anion [CF218FSO2]−, which process comprises treating a difluorocarbene source with (i) a source of 18F− and (ii) a source of SO2.
The invention also provides a compound comprising the anion [CF218FSO2]−.
The invention also provides a process for producing a compound comprising an 18F-trifluoromethyl functionalised aromatic group, which process comprises contacting a compound comprising an aromatic group with a compound comprising the anion [CF218FSO2]− in the presence of an activator for trifluoromethyl radical formation.
The present invention also provides a compound comprising an 18F-trifluoromethyl functionalised aromatic group.
The invention also provides a compound comprising an 18F-trifluoromethyl functionalised aromatic group for use in a method for treatment of the human or animal body by therapy or for use in a diagnostic method practised on the human or animal body.
The invention also provides a method of imaging a subject, comprising administering to the subject a compound comprising an 18F-trifluoromethyl functionalised aromatic group, or a pharmaceutically acceptable salt thereof, and imaging the subject by positron emission tomography (PET).
The term “alkyl group”, as used herein, refers to a substituted or unsubstituted, straight or branched chain saturated hydrocarbon radical. Typically an alkyl group is C1-20 alkyl, or C1-10 alkyl, for example methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl or decyl (including straight or branched chain isomers thereof), or C1-6 alkyl, for example methyl, ethyl, propyl, butyl, pentyl or hexyl (including straight or branched chain isomers thereof), or C1-4 alkyl, for example methyl, ethyl, i-propyl, n-propyl, t-butyl, s-butyl or n-butyl. When an alkyl group is substituted it typically bears one or more substituents selected from substituted or unsubstituted C1-20 alkyl, substituted or unsubstituted C2-20 alkenyl, substituted or unsubstituted C2-20 alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, cyano, amino, C1-10 alkylamino, di(C1-10)alkylamino, arylamino, diarylamino, arylalkylamino, amido, acylamido, hydroxy, oxo, halo, carboxy, ester, acyl, acyloxy, C1-20 alkoxy, aryloxy, haloalkyl, sulfonic acid, sulfhydryl (i.e. thiol, —SH), C1-10 alkylthio, arylthio, sulfonyl, phosphoric acid, phosphate ester, phosphonic acid and phosphonate ester. Examples of substituted alkyl groups include haloalkyl, hydroxyalkyl, aminoalkyl, alkoxyalkyl and alkaryl groups. The term alkaryl, as used herein, pertains to a C1-20 alkyl group in which at least one hydrogen atom has been replaced with an aryl group. Examples of such groups include, but are not limited to, benzyl (phenylmethyl, PhCH2—), benzhydryl (Ph2CH—), trityl (triphenylmethyl, Ph3C—), and phenethyl (phenylethyl, Ph-CH2CH2—). Typically a substituted alkyl group carries 1, 2 or 3 substituents, for instance 1 or 2.
The term “alkenyl”, as used herein, refers to a linear or branched chain hydrocarbon radical comprising one or more double bonds. An alkenyl group may be a C2-20 alkenyl group, a C2-10 alkenyl group or a C2-6 alkenyl group. Examples of C2-20 alkenyl groups include those related to C2-20 alkyl groups by the insertion of one or more double bonds. Alkenyl groups typically comprise one or two double bonds. The alkenyl groups referred to herein may be substituted or unsubstituted, as defined for alkyl groups above.
The term “alkynyl”, as used herein, refers to a linear or branched chain hydrocarbon radical comprising one or more triple bonds. An alkynyl group may be a C2-20 alkynyl group, a C2-10 alkynyl group a C2-6 alkynyl group. Examples of C2-20 alkynyl groups include those related to C2-20 alkyl groups by the insertion of one or more triple bonds. Alkynyl groups typically comprise one or two triple bonds. The alkynyl groups referred to herein may be substituted or unsubstituted, as defined for alkyl groups above.
The term “cycloalkyl group”, as used herein, refers to a substituted or unsubstituted alkyl group which is also a cyclyl group; that is, a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a carbocyclic ring of a carbocyclic compound. A cycloalkyl group may have from 3 to 25 carbon atoms (unless otherwise specified), including from 3 to 25 ring atoms. Thus, the term “cycloalkyl” includes the sub-classes cycloalkyenyl and cycloalkynyl. Examples of groups of C3-25 cycloalkyl groups include C3-20 cycloalkyl, C3-15 cycloalkyl, C3-10 cycloalkyl, and C3-25 cycloalkyl. When a C3-25 cycloalkyl group is substituted it typically bears one or more substituents selected from substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, cyano, amino, C1-10 alkylamino, di(C1-10)alkylamino, arylamino, diarylamino, arylalkylamino, amido, acylamido, hydroxy, oxo, halo, carboxy, ester, acyl, acyloxy, C1-20 alkoxy, aryloxy, haloalkyl, sulfonic acid, sulfhydryl (i.e. thiol, —SH), C1-10 alkylthio, arylthio, phosphoric acid, phosphate ester, phosphonic acid and phosphonate ester and sulfonyl. Typically a substituted cycloalkyl group carries 1, 2 or 3 substituents, for instance 1 or 2.
Examples of C3-25 cycloalkyl groups include, but are not limited to, those derived from saturated monocyclic hydrocarbon compounds, which C3-25 cycloalkyl groups are substituted or unsubstituted as defined above: cyclopropane (C3), cyclobutane (C4), cyclopentane (C5), cyclohexane (C6), cycloheptane (C7), methylcyclopropane (C4), dimethylcyclopropane (C5), methylcyclobutane (C5), dimethylcyclobutane (C6), methylcyclopentane (C6), dimethylcyclopentane (C7), methylcyclohexane (C7), dimethylcyclohexane (C8), menthane (C10); unsaturated monocyclic hydrocarbon compounds: cyclopropene (C3), cyclobutene (C4), cyclopentene (C5), cyclohexene (C6), methylcyclopropene (C4), dimethylcyclopropene (C5), methylcyclobutene (C5), dimethylcyclobutene (C6), methylcyclopentene (C6), dimethylcyclopentene (C7), methylcyclohexene (C7), dimethylcyclohexene (C8); saturated polycyclic hydrocarbon compounds: thujane (C10), carane (C10), pinane (C10), bornane (C10), norcarane (C7), norpinane (C7), norbornane (C7), adamantane (C10), decalin (decahydronaphthalene) (C10); unsaturated polycyclic hydrocarbon compounds: camphene (C10), limonene (C10), pinene (C10); polycyclic hydrocarbon compounds having an aromatic ring: indene (C9), indane (e.g., 2,3-dihydro-1H-indene) (C9), tetraline (1,2,3,4-tetrahydronaphthalene) (C10), acenaphthene (C12), fluorene (C13), phenalene (C13), acephenanthrene (C15), aceanthrene (C16), cholanthrene (C20).
The term “heterocyclyl group”, as used herein, refers to a substituted or unsubstituted monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound, which moiety has from 3 to 20 ring atoms (unless otherwise specified), of which from 1 to 10 are ring heteroatoms. Heterocyclic compounds include aromatic heterocyclic compounds and non-aromatic heterocyclic compounds. Preferably, each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ring heteroatoms. When a C3-20 heterocyclyl group is substituted it typically bears one or more substituents selected from C1-6 alkyl which is unsubstituted, aryl (as defined herein), cyano, amino, C1-10 alkylamino, di(C1-10)alkylamino, arylamino, diarylamino, arylalkylamino, amido, acylamido, hydroxy, oxo, halo, carboxy, ester, acyl, acyloxy, C1-20 alkoxy, aryloxy, haloalkyl, sulfonic acid, sulfhydryl (i.e. thiol, —SH), C1-10 alkylthio, arylthio, phosphoric acid, phosphate ester, phosphonic acid and phosphonate ester and sulfonyl. Typically a substituted C3-20 heterocyclyl group carries 1, 2 or 3 substituents, for instance 1 or 2.
Examples of groups of heterocyclyl groups include C3-20 heterocyclyl, C5-20 heterocyclyl, C3-15 heterocyclyl, C5-15 heterocyclyl, C3-12 heterocyclyl, C5-12 heterocyclyl, C3-10 heterocyclyl, C5-10 heterocyclyl, C3-7 heterocyclyl, C5-7 heterocyclyl, and C5-6 heterocyclyl.
Examples of (non-aromatic) monocyclic C3-20 heterocyclyl groups include, but are not limited to, those derived from:
N1: aziridine (C3), azetidine (C4), pyrrolidine (tetrahydropyrrole) (C5), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole) (C5), 2H-pyrrole or 3H-pyrrole (isopyrrole, isoazole) (C5), piperidine (C6), dihydropyridine (C6), tetrahydropyridine (C6), azepine (C7);
O1: oxirane (C3), oxetane (C4), oxolane (tetrahydrofuran) (C5), oxole (dihydrofuran) (C5), oxane (tetrahydropyran) (C6), dihydropyran (C6), pyran (C6), oxepin (C7);
S1: thiirane (C3), thietane (C4), thiolane (tetrahydrothiophene) (C5), thiane (tetrahydrothiopyran) (C6), thiepane (C7);
O2: dioxolane (C5), dioxane (C6), and dioxepane (C7);
O3: trioxane (C6);
N2: imidazolidine (C5), pyrazolidine (diazolidine) (C5), imidazoline (C5), pyrazoline (dihydropyrazole) (C5), piperazine (C6);
N1O1: tetrahydrooxazole (C5), dihydrooxazole (C5), tetrahydroisoxazole (C5), dihydroisoxazole (C5), morpholine (C6), tetrahydrooxazine (C6), dihydrooxazine (C6), oxazine (C6);
N1S1: thiazoline (C5), thiazolidine (C5), thiomorpholine (C6);
N2O1: oxadiazine (C6);
O1S1: oxathiole (C5) and oxathiane (thioxane) (C6); and,
N1O1S1: oxathiazine (CQ).
Examples of substituted (non-aromatic) monocyclic heterocyclyl groups include those derived from saccharides, in cyclic form, for example, furanoses (C5), such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse, and pyranoses (C6), such as allopyranose, altropyranose, glucopyranose, mannopyranose, gulopyranose, idopyranose, galactopyranose, and talopyranose.
Examples of C3-20 heterocyclyl groups which are also aryl groups are described below as heteroaryl groups.
The term “aryl group”, as used herein, refers to a substituted or unsubstituted, monocyclic or polycyclic (for instance bicyclic) aromatic group which typically contains from 6 to 14 carbon atoms, preferably from 6 to 10 carbon atoms in the ring portion. Examples include phenyl, naphthyl, indenyl, indanyl, anthracenyl and pyrenyl groups. An aryl group is substituted or unsubstituted. When an aryl group is substituted it typically bears one or more substituents selected from substituted or unsubstituted C1-20 alkyl, substituted or unsubstituted C2-20 alkenyl, substituted or unsubstituted C2-20 alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, cyano, amino, C1-10 alkylamino, di(C1-10)alkylamino, arylamino, diarylamino, arylalkylamino, amido, acylamido, hydroxy, oxo, halo, carboxy, ester, acyl, acyloxy, C1-20 alkoxy, aryloxy, haloalkyl, sulfonic acid, sulfhydryl (i.e. thiol, —SH), C1-10 alkylthio, arylthio, sulfonyl, phosphoric acid, phosphate ester, phosphonic acid and phosphonate ester. Typically it carries 0, 1, 2 or 3 substituents. A substituted aryl group may be substituted in two positions with a single C1-6 alkylene group, or with a bidentate group represented by the formula —X—C1-6 alkylene, or —X—C1-6 alkylene-X—, wherein X is selected from O, S and NR, and wherein R is H, aryl or C1-6 alkyl. Thus a substituted aryl group may be an aryl group fused with a cycloalkyl group or with a heterocyclyl group. The term “aralkyl” as used herein, pertains to an aryl group in which at least one hydrogen atom (e.g., 1, 2, 3) has been substituted with a C1-6 alkyl group. Examples of such groups include, but are not limited to, tolyl (from toluene), xylyl (from xylene), mesityl (from mesitylene), and cumenyl (or cumyl, from cumene), and duryl (from durene).
The ring atoms of an aryl group may include one or more heteroatoms (as in a heteroaryl group). Such an aryl group is a heteroaryl group, and is a substituted or unsubstituted monocyclic or polycyclic (for instance bicyclic) heteroaromatic group which typically contains from 6 to 14 atoms, for instance 6 to 10 atoms, in the ring portion including one or more heteroatoms. It is generally a 5- or 6-membered ring, containing at least one heteroatom selected from O, S, N, P, Se and Si. It may contain, for example, 1, 2 or 3 heteroatoms. Examples of heteroaryl groups include pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, furanyl, thienyl, pyrazolidinyl, pyrrolyl, oxazolyl, oxadiazolyl, isoxazolyl, thiadiazolyl, thiazolyl, isothiazolyl, imidazolyl, pyrazolyl, triazolyl, indolyl, benzimidazolyl, indazolyl, benzotriazolyl, pyrrolopyridinyl, pyrrolopyrimidinyl, purinyl, indolizinyl, pyrrolopyrazinyl, pyrrolopyriminyl, pyrrolopyridazinyl, imidazopyridinyl, pyrazolopyridinyl, imidazopyridazinyl, imidazopyrimidinyl, imidazopyrazinyl, imidazopyrimidinyl, triazolopyridinyl, quinolyl, isoquinolyl, cinnolinyl, quinazolinyl, quinoxalinyl, phthalazinyl, pyridopyrazinyl, pteridinyl, pyridopyridazinyl, naphthyridinyl, and carbazolyl. A heteroaryl group may be substituted or unsubstituted, for instance, as specified above for aryl. Typically it carries 0, 1, 2 or 3 substituents.
The term “alkylene group” as used herein, refers to an substituted or unsubstituted bidentate moiety obtained by removing two hydrogen atoms, either both from the same carbon atom, or one from each of two different carbon atoms, of a hydrocarbon compound having from 1 to 20 carbon atoms (unless otherwise specified), which may be aliphatic or alicyclic, and which may be saturated, partially unsaturated, or fully unsaturated. Thus, the term “alkylene” includes the sub-classes alkenylene, alkynylene, cycloalkylene, etc., discussed below. Typically it is C1-10 alkylene, for instance C1-6 alkylene. Typically it is C1-4 alkylene, for example methylene, ethylene, i-propylene, n-propylene, t-butylene, s-butylene or n-butylene. It may also be pentylene, hexylene, heptylene, octylene and the various branched chain isomers thereof. An alkylene group may be substituted or unsubstituted, for instance, as specified above for alkyl. Typically a substituted alkylene group carries 1, 2 or 3 substituents, for instance 1 or 2.
In this context, the prefixes (e.g., C1-4, C1-7, C1-20, C2-7, C3-7, etc.) denote the number of carbon atoms, or range of number of carbon atoms. For example, the term “C1-4alkylene,” as used herein, pertains to an alkylene group having from 1 to 4 carbon atoms. Examples of groups of alkylene groups include C1-4 alkylene (“lower alkylene”), C1-7 alkylene, C1-10 alkylene and C1-20 alkylene.
Examples of linear saturated C1-7 alkylene groups include, but are not limited to, —(CH2)n— where n is an integer from 1 to 7, for example, —CH2— (methylene), —CH2CH2— (ethylene), —CH2CH2CH2— (propylene), and —CH2CH2CH2CH2— (butylene).
Examples of branched saturated C1-7 alkylene groups include, but are not limited to, —CH(CH3)—, —CH(CH3)CH2—, —CH(CH3)CH2CH2—, —CH(CH3)CH2CH2CH2—, —CH2CH(CH3) CH2—, —CH2CH(CH3)CH2CH2—, —CH(CH2CH3)—, —CH(CH2CH3)CH2—, and —CH2CH(CH2CH3)CH2—.
Examples of linear partially unsaturated C1-7 alkylene groups include, but are not limited to, —CH═CH— (vinylene), —CH═CH—CH2—, —CH2—CH═CH2—, —CH═CH—CH2—CH2—, —CH═CH—CH2—CH2—CH2—, —CH═CH—CH═CH—, —CH—CH—CH—CH—CH2—, —CH—CH—CH—CH—CH2—CH2—, —CH—CH—CH2—CH—CH—, and —CH═CH—CH2—CH2—CH═CH—.
Examples of branched partially unsaturated C1-7 alkylene groups include, but are not limited to, —C(CH3)═CH—, —C(CH3)═CH—CH2—, and —CH═CH—CH(CH3)—.
Partially unsaturated alkylene groups comprising one or more double bonds may be referred to as alkenylene groups. Partially unsaturated alkylene groups comprising one or more triple bonds may be referred to as alkynylene groups (for instance —C≡C—, CH2—C≡C—, and —CH2-C≡C≡CH2—).
Examples of alicyclic saturated C1-7 alkylene groups include, but are not limited to, cyclopentylene (e.g., cyclopent-1,3-ylene), and cyclohexylene (e.g., cyclohex-1,4-ylene). Examples of alicyclic partially unsaturated C1-7 alkylene groups include, but are not limited to, cyclopentenylene (e.g., 4-cyclopenten-1,3-ylene), cyclohexenylene (e.g., 2-cyclohexen-1,4-ylene; 3-cyclohexen-1,2-ylene; 2,5-cyclohexadien-1,4-ylene).
As used herein the term “oxo” represents a group of formula: ═O.
As used herein the term “acyl” represents a group of formula: —C(═O)R, wherein R is an acyl substituent, for example, a substituted or unsubstituted C1-20 alkyl group, substituted or unsubstituted C2-20 alkenyl group, substituted or unsubstituted C2-20 alkynyl group, a substituted or unsubstituted C3-20 heterocyclyl group, a substituted or unsubstituted aryl group or a substituted or unsubstituted heteroaryl group, for instance a substituted or unsubstituted C1-6alkyl group. Examples of acyl groups include, but are not limited to, —C(═O)CH3 (acetyl), —C(═O)CH2CH3 (propionyl), —C(═O)C(CH3)3 (t-butyryl), and —C(═O)Ph (benzoyl, phenone).
As used herein the term “acyloxy” (or reverse ester) represents a group of formula: —OC(═O)R, wherein R is an acyloxy substituent, for example, a substituted or unsubstituted C1-20 alkyl group, substituted or unsubstituted C2-20 alkenyl group, substituted or unsubstituted C2-20 alkynyl group, a substituted or unsubstituted C3-20 heterocyclyl group, a substituted or unsubstituted aryl group or a substituted or unsubstituted heteroaryl group, for instance a substituted or unsubstituted C1-6 alkyl group. Examples of acyloxy groups include, but are not limited to, —OC(═O)CH3 (acetoxy), —OC(═O)CH2CH3, —OC(═O)C(CH3)3, —OC(═O)Ph, and —OC(═O)CH2Ph.
As used herein the term “ester” (or carboxylate, carboxylic acid ester or oxycarbonyl) represents a group of formula: —C(═O)OR, wherein R is an ester substituent, for example, a substituted or unsubstituted C1-20 alkyl group, substituted or unsubstituted C2-20 alkenyl group, substituted or unsubstituted C2-20 alkynyl group, a substituted or unsubstituted C3-20 heterocyclyl group, a substituted or unsubstituted aryl group or a substituted or unsubstituted heteroaryl group, for instance a substituted or unsubstituted C1.6 alkyl group. Examples of ester groups include, but are not limited to, —C(═O)OCH3, —C(═O)OCH2CH3, —C(═O)OC(CH3)3, and —C(═O)OPh.
As used herein the term “amino” represents a group of formula —NH2. The term “C1-C10 alkylamino” represents a group of formula —NHR′ wherein R′ is a C1-10 alkyl group, preferably a C1-6 alkyl group, as defined previously. The term “di(C1-10)alkylamino” represents a group of formula —NR′R″ wherein R′ and R″ are the same or different and represent C1-10 alkyl groups, preferably C1-6 alkyl groups, as defined previously. The term “arylamino” represents a group of formula —NHR′ wherein R′ is an aryl group, preferably a phenyl group, as defined previously. The term “diarylamino” represents a group of formula —NR′R″ wherein R′ and R″ are the same or different and represent aryl groups, preferably phenyl groups, as defined previously. The term “arylalkylamino” represents a group of formula —NR′R″ wherein R′ is a C1-10 alkyl group, preferably a C1-6 alkyl group, and R″ is an aryl group, preferably a phenyl group.
A halo group is chlorine, fluorine, bromine or iodine (a chloro group, a fluoro group, a bromo group or an iodo group). It is typically chlorine, fluorine or bromine.
The term “halide”, as used herein, refers to fluoride, chloride, bromide and iodide.
As used herein the term “amido” represents a group of formula: —C(═O)NR′R″, wherein R′ and R″ are independently amino substituents, as defined for di(C1-10)alkylamino groups. Examples of amido groups include, but are not limited to, —C(═O)NH2, —C(═O)NHCH3, —C(═O)N(CH3)2, —C(═O)NHCH2CH3, and —C(═O)N(CH2CH3)2, as well as amido groups in which R′ and R″, together with the nitrogen atom to which they are attached, form a heterocyclic structure as in, for example, piperidinocarbonyl, morpholinocarbonyl, thiomorpholinocarbonyl, and piperazinocarbonyl.
As used herein the term “acylamido” represents a group of formula: —NR′C(—O)R″, wherein R′ is an amide substituent, for example, hydrogen, a C1-20 alkyl group, a C3-20 heterocyclyl group, an aryl group, preferably hydrogen or a C1-20 alkyl group, and R″ is an acyl substituent, for example, a C1-20 alkyl group, a C3-20 heterocyclyl group, or an aryl group, preferably hydrogen or a C1-20 alkyl group. Examples of acylamide groups include, but are not limited to, —NHC(═O)CH3, —NHC(═O)CH2CH3, —NHC(═O)Ph, —NHC(═O)C15H31 and —NHC(═O)C9H19. Thus, a substituted C1-20 alkyl group may comprise an acylamido substituent defined by the formula —NHC(═O)—C1-20 alkyl, such as —NHC(═O)C15H31 or —NHC(═O)C9H19. R1 and R2 may together form a cyclic structure, as in, for example, succinimidyl, maleimidyl, and phthalimidyl:
A C1-10 alkylthio group is a said C1-10 alkyl group, preferably a C1-6 alkyl group, attached to a thio group. An arylthio group is an aryl group, preferably a phenyl group, attached to a thio group.
A C1-20 alkoxy group is a said substituted or unsubstituted C1-20 alkyl group attached to an oxygen atom. A C1-6 alkoxy group is a said substituted or unsubstituted C1-6 alkyl group attached to an oxygen atom. A C1-4 alkoxy group is a substituted or unsubstituted C1-4 alkyl group attached to an oxygen atom. Examples of C1-4 alkoxy groups include, —OMe (methoxy), —OEt (ethoxy), —O(nPr) (n-propoxy), —O(iPr) (isopropoxy), —O(nBu) (n-butoxy), —O(sBu) (sec-butoxy), —O(iBu) (isobutoxy), and —O(tBu) (tert-butoxy). Further examples of C1-20 alkoxy groups are —O(Adamantyl), —O—CH2-Adamantyl and —O—CH2—CH2-Adamantyl.
An aryloxy group is a substituted or unsubstituted aryl group, as defined herein, attached to an oxygen atom. An example of an aryloxy group is —OPh (phenoxy).
Unless otherwise specified, included in the above are the well known ionic, salt, solvate, and protected forms of these substituents. For example, a reference to carboxylic acid or carboxyl group (—COOH) also includes the anionic (carboxylate) form (—COO−), a salt or solvate thereof, as well as conventional protected forms. Similarly, a reference to an amino group includes the protonated form (—N+HR′R″), a salt or solvate of the amino group, for example, a hydrochloride salt, as well as conventional protected forms of an amino group. Similarly, a reference to a hydroxyl group also includes the anionic form (—O−), a salt or solvate thereof, as well as conventional protected forms.
Certain compounds may exist in one or more particular geometric, optical, enantiomeric, diastereomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r-forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d- and l-forms; (+) and (−) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; α- and β-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referred to as “isomers” (or “isomeric forms”).
Note that, except as discussed below for tautomeric forms, specifically excluded from the term “isomers,” as used herein, are structural (or constitutional) isomers (i.e., isomers which differ in the connections between atoms rather than merely by the position of atoms in space). For example, a reference to a methoxy group, —OCH3, is not to be construed as a reference to its structural isomer, a hydroxymethyl group, —CH2OH. Similarly, a reference to ortho-chlorophenyl is not to be construed as a reference to its structural isomer, meta-chlorophenyl. However, a reference to a class of structures may well include structurally isomeric forms falling within that class (e.g., C1-7 alkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).
The above exclusion does not pertain to tautomeric forms, for example, keto, enol, and enolate forms, as in, for example, the following tautomeric pairs: keto/enol, imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime, thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.
Note that specifically included in the term “isomer” are compounds with one or more isotopic substitutions. For example, H may be in any isotopic form, including 1H, 2H (D), and 3H (T); C may be in any isotopic form, including 12C, 13C, and 14C; O may be in any isotopic form, including 16O and 18O; and the like, unless otherwise specified. However, reference to an isotope of fluorine refers only to that isotope of fluorine. In particular, reference to 18F includes only 18F. Reference to fluorine without specifying the isotope may refer to 18F or 19F depending on context. Typically, reference to “F” (i.e. without defining the isotope) refers to the 19F, i.e. stable fluorine.
Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including (wholly or partially) racemic and other mixtures thereof. Methods for the preparation (e.g., asymmetric synthesis) and separation (e.g., fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting known methods, in a known manner.
The term “substituted”, as used herein, may be as defined above for particular groups. However, in some instances, the term substituted may refer to a group substituted with a group selected from substituted or unsubstituted C1-20 alkyl, substituted or unsubstituted C2-20 alkenyl, substituted or unsubstituted C2-20 alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, cyano, amino, C1-10 alkylamino, di(C1-10)alkylamino, arylamino, diarylamino, arylalkylamino, amido, acylamido, hydroxy, oxo, halo, carboxy, ester, acyl, acyloxy, C1-20 alkoxy, aryloxy, haloalkyl, sulfonic acid, sulfhydryl (i.e. thiol, —SH), C1-10 alkylthio, arylthio, sulfonyl, phosphoric acid, phosphate ester, phosphonic acid and phosphonate ester. In other instances, the term “substituted” may refer to a group substituted with a group selected from substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C2-6 alkenyl, substituted or unsubstituted C2-6 alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, cyano, amino, C1-6 alkylamino, di(C1-6)alkylamino, arylamino, diarylamino, arylalkylamino, amido, acylamido, hydroxy, oxo, halo, carboxy, ester, acyl, acyloxy, C1-6 alkoxy, aryloxy, haloalkyl, sulfonic acid, thiol, C1-6 alkylthio, arylthio, sulfonyl, phosphoric acid, phosphate ester, phosphonic acid and phosphonate ester. For example, the term “substituted” may refer to a group substituted with a group selected unsubstituted C1-6 alkyl, unsubstituted C2-6 alkenyl, unsubstituted C2-6 alkynyl, unsubstituted aryl, unsubstituted heteroaryl, cyano, amino, unsubstituted C1-6 alkylamino, unsubstituted di(C1-6)alkylamino, unsubstituted arylamino, unsubstituted diarylamino, unsubstituted arylalkylamino, unsubstituted amido, unsubstituted acylamido, hydroxy, oxo, halo, carboxy, unsubstituted ester, unsubstituted acyl, unsubstituted acyloxy, unsubstituted C1-6 alkoxy, unsubstituted aryloxy, sulfonic acid, thiol, unsubstituted C1-6 alkylthio, unsubstituted arylthio, sulfonyl, phosphoric acid, unsubstituted phosphate ester, unsubstituted phosphonic acid and unsubstituted phosphonate ester.
The term “18F” refers to an atom of the specific isotope of fluorine having 9 protons and 9 neutrons. The terms “18F−” and “18F-fluoride” refer to an anion of the atom of the specific isotope of fluorine having 9 protons and 9 neutrons.
The use of “18F-” before a chemical entity name or “18F[chemical formula]” refers to a chemical entity in which a 19F has been replaced with an 18F. Therefore, the terms “18F-trifluoromethyl” and “18F[CF3]” as used herein refer to a —CF3 (trifluoromethyl) group in which one of the three fluorines is 18F, i.e. a group of formula —CF218F.
The term “leaving group” as used herein refers to an atom or group, either charged or uncharged, that becomes detached from an atom in the residual or main part of the substrate in a particular reaction. The leaving group may or may not retain the bonding pair of electrons when it detaches from the atom in the residual or main part of the substrate. After detaching from the atom in the residual or main part of the substrate, the leaving group may have a positive charge, a negative charge or no charge (neutral charge).
The term “ligand”, as used herein, refers to a species capable of binding to a central atom to form a complex. Ligands may be charged or neutral species. Typically, as referred to herein, a ligand is a neutral species.
The term “transition metal” as used herein means any one of the three series of elements arising from the filling of the 3d, 4d and 5d shells, and situated in the periodic table following the alkaline earth metals. This definition is used in N. N. Greenwood and A. Earnshaw “Chemistry of the Elements”, First Edition 1984, Pergamon Press Ltd., at page 1060, first paragraph, with respect to the term “transition element”. The same definition is used herein for the term “transition metal”. Thus, the term “transition metal”, as used herein, includes all of Sc, Y, Ti, Zr, Hf, V, Nb, Ta, Cr, Mo, W, Mn, Tc, Re, Fe, Ru, Os, Co, Rh, Ir, Ni, Pd, Pt, Cu, Ag, Au, Zn, Cd and Hg. These are also referred to as the first, second and third row transition metals (i.e. the transition metals in periods 4, 5 and 6 of the periodic table).
The term “carbene” as used herein refers to electrically neutral species H2C: and its derivatives, in which the carbon is covalently bonded to two univalent groups of any kind or a divalent group. The carbon of the carbene bears two nonbonding electrons, which may be spin-paired (singlet state) or spin-non-paired (triplet state). The term “difluorocarbene” as used herein refers to F2C.
Process
The invention provides a process for producing a compound comprising the anion [CF218FSO2]−, which process comprises treating a difluorocarbene source with
Typically the difluorocarbene source is treated with both (i) and (ii) simultaneously. Thus, typically the process comprises treating the difluorocarbene source with 18F− in the presence of the source of SO2. Equivalently, the process may comprise treating the difluorocarbene source with the source of SO2 in the presence of 18F−. The source of 18F− may be any suitable source of 18F−, as discussed below. Often 18F− will be solvated.
Difluorocarbene Source
Any suitable difluorocarbene source may be used in the process of the invention. Typically, the difluorocarbene source provides difluorocarbene via an alpha elimination reaction. The alpha elimination reaction is usually a transformation of the type:
wherein R1 is a first leaving group and R2 is a second leaving group. The loss of R1 and R2 from the central CF2 group generates difluorocarbene (:CF2). Typically, the difluorocarbene generated is free difluorocarbene.
The difluorocarbene source may be a compound of Formula (I):
wherein R1 is a first leaving group and R2 is a second leaving group. R1 generally retains the (CF2—R1) bonding electron pair when R1 detaches from the central CF2 moiety. R2 usually does not retain the (CF2—R2) bonding electron pair when R2 detaches from the central CF2 moiety.
R1 may comprise a positively charged functional group. R2 may comprise a negatively charged functional group. One or both of R1 and R2 may have no charge (neutral charge). In the case where there is a net charge on the compound of Formula I, one or more counterions may be present. For instance, if the compound of Formula I is positively charged, one or more counter-anions may be present. The one or more counter-anions may be selected from any anion described herein. For instance, the one or more anions may be selected from halide, hydroxide, sulfate, and nitrate. Alternatively, the compound of Formula I may be anionic, and associated with one or more counter-cations. Again, any suitable counter-cation may be employed; many such cations are known to the skilled person.
In some instances, R1 may comprise a positively charged functional group and R2 may comprise a negatively charged functional group. In this case, there may be no net charge on the compound of Formula I. Thus, the compound of Formula I may be zwitterionic.
Examples of suitable R1 groups include:
Examples of suitable R2 groups include:
In some instances, R1 and R2, together with the CF2 group to which they are attached may form a cyclic structure.
Examples of difluorocarbene sources are set out in the review by Ni et al. (Ni. C., Hu. J., Recent Advances in the Synthetic Application ofDifluorocarbene, SYNTHESIS, 2014, 46, 0842-0863). For instance, the difluorocarbene source may be CF3H, HCF2Cl, HCF2Br, ClCF2C(O)O−Na+, ClCF2C(O)OMe, BrCF2C(O)O−Na+, FSO2CF2COOH, FS(O)2CF2C(O)OMe, F3CS(O)2CF2H, FS(O)2CF2C(O)OSi(CH3)3 (TFDA), ClF2CSi(CH3)3, BrF2CSi(CH3)3, F3CSi(CH3)3, ClF2CC(O)Ph, ClF2CS(O)2Ph, BrF2CP(O)(OEt)2, HF2CS(O)(NTs)Ph, HF2COTf, [PhArSCF2Br]−+OTf−, [HF2CN(nBu)3]+Cl−, Ph3P+CF2C(O)O−, HgICF3, (CH3)3SnCF3 or tetrafluoroethane beta-sulfone derivatives.
Typically, R1 is a phosphonium or an ammonium cation. For instance, R1 may be a phosphonium cation of formula —[PR3R4R5]+ or an ammonium cation of formula —[NR3R4R5]+, wherein R3, R4 and R5 are each independently selected from H, substituted or unsubstituted C1-20 alkyl, substituted or unsubstituted C2-20 alkenyl, substituted or unsubstituted C2-20 alkynyl, substituted or unsubstituted C3-20 cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl; and wherein two or more of R3, R4 and R5 may be bonded together to form one or more rings.
Preferably, R1 is a phosphonium cation of formula —[PR3R4R5]+. For instance, R1 may be a phosphonium cation of formula —[PR3R4R5]+ wherein R3, R4 and R5 are substituted or unsubstituted aryl. Thus, R1 may be —[PPh3]+.
Typically, R2 is —C(O)O−, or a group of formula —C(O)OR9, wherein R9 is selected from hydrogen, substituted or unsubstituted C1-20 alkyl, substituted or unsubstituted C2-20 alkenyl, substituted or unsubstituted C2-20 alkynyl, substituted or unsubstituted C3-20 cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl; or wherein R9 is a group of formula —Si(R10R11R12) wherein R10, R11 and R12 are each independently selected from hydrogen, substituted or unsubstituted C1-20 alkyl, substituted or unsubstituted C2-20 alkenyl, substituted or unsubstituted C2-20 alkynyl, substituted or unsubstituted C3-20 cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, C1-20 alkoxy, aryloxy and halo. Preferably, R2 is —C(O)O−.
Thus, in Formula I, the R1 group may be a phosphonium or ammonium cation and the R2 group may be —C(O)O−, or a group of formula —C(O)OR9, wherein R9 is selected from hydrogen, substituted or unsubstituted C1-20 alkyl, substituted or unsubstituted C2-20 alkenyl, substituted or unsubstituted C2-20 alkynyl, substituted or unsubstituted C3-20 cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl; or wherein R9 is a group of formula —Si(R10R11R12) wherein R10, R11 and R12 are each independently selected from hydrogen, substituted or unsubstituted C1-20 alkyl, substituted or unsubstituted C2-20 alkenyl, substituted or unsubstituted C2-20 alkynyl, substituted or unsubstituted C3-20 cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, C1-20 alkoxy, aryloxy and halo.
Preferably R1 is a phosphonium cation of formula —[PR3R4R5]+, wherein R3, R4 and R5 are each independently selected from H, substituted or unsubstituted C1-20 alkyl, substituted or unsubstituted C2-20 alkenyl, substituted or unsubstituted C2-20 alkynyl, substituted or unsubstituted C3-20 cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl; and wherein two or more of R3, R4 and R5 may be bonded together to form one or more rings, and R2 is —C(O)O−.
Thus, in one embodiment the difluorocarbene source is (triphenylphosponio)difluoroacetate ([Ph3P]+CF2COO−).
The generation of difluorocarbene from the difluorocarbene source may require an additive to initiate difluorocarbene generation. Typically, the additive is a nucleophile or a base. Examples of nucleophiles include, but are not limited to halide or hydroxide. Examples of bases include, but are not limited to metal hydroxides, metal carbonates, metal hydrides, metal alkoxides and phosphinimine bases.
The difluorocarbene source may release difluorcarbene as a result of heating (i.e. via thermal pyrolysis). Typically, the difluorocarbene source is heated to a temperature from 80 to 150° C. For instance, the difluorocarbene source may be (triphenylphosponio)difluoroacetate (Ph3P+CF2C(O)O−) and the process of the invention may comprise heating the difluorocarbene source to a temperature of from 80 to 150° C. to generate difluorcarbene.
Source of SO2
Any suitable source of SO2 may be used in the process of the invention. The source of SO2 may be free SO2 which is a gas at room temperature, or a compound that provides SO2 in situ. Typically, the source of SO2 is a compound that provides SO2 in situ. Typically the compound that provides SO2 in situ comprises a heteroatom-SO2 bond, for instance an N—SO2 bond.
Thus, the source of SO2 may be a compound of Formula (II):
Typically, R6 is selected from substituted or unsubstituted C1-20 alkyl, substituted or unsubstituted C2-20 alkenyl, substituted or unsubstituted C2-20 alkynyl, substituted or unsubstituted C3-20 cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl; and R7 and R8 are substituted or unsubstituted C1-20 alkyl groups which are bonded to a single heteroatom to form a ring, optionally wherein the heteroatom is O, S or N, wherein said N may be part of a group NRy or N+RyRz wherein Ry is H, C1-6 alkyl or aryl, and R is SO2. Often R6 is substituted or unsubstituted C1-20 alkyl. Usually the heteroatom is O.
In one embodiment, the source of SO2 is a compound of Formula (III):
In one embodiment, the source of SO2 is a compound of Formula (IV):
Compounds of Formulae II, III and IV advantageously act as sources of SO2 without the need for employing toxic gaseous reagents. Further, N-methylmorpholine-SO2 is particularly advantageous because the side products when this reagent is used are volatile and easy to separate out from the reaction mixture following reaction, for instance during purification.
18F− Source
The 18F− used in the process of the invention may be in any suitable form. Typically, the 18F− is present as a salt. Thus, the source of 18F− may be, or may comprise, a salt of 18F−. Thus, the process of the invention may comprise treating the difluorocarbene source with (i) a salt of 18F− and (ii) the source of SO2. Typically the concentration of 18F− is less than or equal to 10−4 M, for instance less than or equal to 10−5 M. In some cases, the concentration of 18F− will be nanomolar or less, for instance less than or equal to 10−8 M. 19F− may also be present. In such a case, the total fluoride concentration (including 18F− and 19F−) may be less than or equal to 10−4 M, for instance less than or equal to 10−5 M.
Any suitable source of 18F− may be used. As will be understood by the skilled person the 18F− will typically be present in the form of a salt, with a counter cation. Any suitable counter cation may be used. Typically, the counter cation is a quaternary ammonium cation, for instance tetrabutylammonium, or an alkali metal cation, for instance Cs+ or K+, or a proton, H+. Preferably, the source of 18F− comprises an alkali metal or ammonium salt of 18F−.
When an alkali metal cation is employed, the alkali metal cation may be complexed in a cryptand, for instance aminopolyether 2.2.2 (K222), which is commercially available as Kryptofix-222. Thus, the source of 18F− may further comprise a cryptand ligand. Advantageously, the addition of such a cryptand enables the fluoride ion 18F− to be solubilized in a polar aprotic solvent, for instance acetonitrile or DMF. It also enables the formation of a ‘naked fluoride ion’ as a KF-K222 complex. In one embodiment, therefore, the source of 18F− is a K[18F]F-K222 complex. Alternatively, the source of 18F− may be [18F]TEAF (tetraethylammonium fluoride), [18F]TBAF (tetrabutylammonium fluoride), [18F]CsF, or [18F]HF. Typically, 18F− is present as K[18F]F-K222 or [18F]HF. More typically, 18F− is present as K[18F]F-K222.
The process of the present invention typically comprises treating the difluorocarbene source with at least 2 GBq of the 18F−.
Process—Further Details
Typically, the compound comprising [CF218FSO2]− further comprises a counter-cation. Thus, the process of the present invention may be a process for producing a compound of formula [CF218FSO2]−nAn+, wherein n is an integer of from 1 to 4.
Typically n is 1 or 2. Preferably, n is 1. When n is 1, An+ may, for instance, be an alkali metal cation or an ammonium cation. Thus, An+ may be Li+, Na+, K+, Rb+, Cs+ or Fr+. Ammonium cations include groups of formula [NRaRbRcRd]+, wherein Ra, Rb, Rc and Rd are each independently selected from H, substituted or unsubstituted C1-20 alkyl, substituted or unsubstituted C2-20 alkenyl, substituted or unsubstituted C2-20 alkynyl, substituted or unsubstituted C3-20 cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl; and wherein two or more of Ra, Rb, Rc and Rd may be bonded together to form one or more rings. Typically, Ra, Rb, Rc and Rd are each independently selected from hydrogen and unsubstituted C1-20 alkyl. Preferably, the ammonium cation is [NH4]+. Therefore, the process may be a process for producing CF218FSO2NH4.
When n is 2, An+ may be a divalent metal cation. For instance, A may be a divalent metal cation selected from alkaline earth metal cations and divalent transition metal cations. Such divalent metal cations include, but are not limited to Mg2+, Ca2+, Sr2+, Ba2+, Sc2+, Ti2+, V2+, Cr2+, Mn2+, Fe2+, Ni2+, Cu2+ or Zn2+.
In one embodiment of the process, the step of treating the difluorocarbene source with the source of 18F− and the source of SO2 is performed in the presence of An+. In this embodiment, the source of 18F− may comprise a metal salt of 18F−. The source of 18F− may comprise a metal salt of 18F−, wherein An+ is said metal. Preferably, the source of 18F− comprises an alkali metal salt of 18F−, wherein An+ is said alkali metal. Therefore, the step of treating the difluorocarbene source with the source of 18F− and the source of SO2 may be performed in the presence of An+, wherein the source of 18F− comprises an A salt of 18F−, preferably wherein An+ is an alkali metal. Therefore, the process may be a process for producing an alkali metal salt of [CF218FSO2], for instance CF218FSO2K.
In another embodiment of the process, the step of treating the difluorocarbene source with the source of 18F− and the source of SO2 is performed in the presence of a first cation Bm+ to produce a compound of formula [CF218FSO2]−mBm+, wherein m is an integer of from 1 to 4, and the process further comprises replacing the first cation Bm+ with a different cation An+, to produce said compound of formula [CF218FSO2]−nAn+.
In this embodiment, the source of 18F− may comprise a metal salt of 18F−, wherein Bm+ is said metal. Preferably the source of 18F− comprises an alkali metal salt of 18F−, wherein Bm+ is said alkali metal. Thus, Bm+ may be Li+, Na+, K+, Rb+, Cs+ or Fr+. The compound of formula [CF218FSO2]−mBm+ is often CF218FSO2K.
In this embodiment, An+ may be a non-metal cation. Preferably, An+ is an ammonium cation. Typically, An+ is an ammonium cation of formula [NRaRbRcRd]+, wherein Ra, Rb, Rc and Rd are each independently selected from H, substituted or unsubstituted C1-20 alkyl, substituted or unsubstituted C2-20 alkenyl, substituted or unsubstituted C2-20 alkynyl, substituted or unsubstituted C3-20 cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl; and wherein two or more of Ra, Rb, Rc and Rd may be bonded together to form one or more rings. Typically, Ra, Rb, Rc and Rd are each independently selected from hydrogen and unsubstituted C1-20 alkyl. Preferably, An+ is [NH4]+. Therefore, the compound of formula [CF218FSO2]−nAn+ may be CF218FSO2NH4.
In this embodiment, the step of replacing the first cation Bm+ with a different cation An+ to produce said compound of formula [CF218FSO2−]nAn+ may be a purification step. The cation An+ may be a cation present in an elution buffer in said purification step. The purification step may be any purification step as described herein, for example the purification step may comprise (i) weak anion exchange chromatography, (ii) chromatography using a mixed-mode, strong anion-exchange cartridge (MAX cartridge) or (iii) reverse phase high performance liquid chromatography (HPLC).
The process of the invention is typically carried out in solution. The process may be carried out in solution in any suitable solvent. Typically the solvent is an aprotic solvent. For instance, the process may be carried out in the presence of a polar aprotic solvent. Typically, the process of the invention is carried out in the presence of one or more aprotic solvents, for instance one or more polar aprotic solvents.
Polar aprotic solvents are well known to the skilled person. The process may be carried out in the presence of a solvent selected from dimethyl formamide, propylene carbonate, N,N-dimethyl acetamide and acetonitrile. The one or more polar aprotic solvents may for instance be selected from dimethylformamide, propylene carbonate and mixtures thereof. For instance, the process may be carried out in a mixture of solvents, such as a mixture of dimethyl formamide and propylene carbonate.
The process may be performed under any suitable atmosphere. For instance, the process may be performed under an inert atmosphere such as nitrogen or argon, or the process may be performed in the presence of oxygen, for instance in air. Often, the process is performed in air.
The amount of the difluorocarbene source may be any suitable amount. The ratio of the amount of the difluorocarbene source to the amount of the SO2 source may be from 1:40 to 40:1, for instance from 1:20 to 20:1. Typically, the amount of SO2 source is less than the amount of difluorocarbene source. The molar ratio of the amount of the difluorocarbene source to the amount of the SO2 source may be from 1:1 to 20:1, preferably 1:1 to 10:1, more preferably 1:1 to 5:1.
The amount of the source of 18F− may be any suitable amount. Typically, the difluorocarbene source is treated with at least 2 GBq of the 18F−. The difluorocarbene source may be treated with at least 3 GBq of the 18F−, at least 4 GBq of the 18F−, at least 5 GBq of the 18F−, at least 6 GBq of the 18F−, at least 7 GBq of the 18F−, at least 8 GBq of the 18F−, at least 9 GBq of the 18F− or at least 10 GBq of the 18F−.
In some embodiments, the difluorocarbene source, the 18F− source and the SO2 source are heated to a temperature of greater than room temperature, for instance to a temperature of at least 50° C., or a temperature of at least 70° C., for example a temperature of at least 80° C., such as, for instance, a temperature of from 80 to 150° C. Such a temperature may be used to induce thermal pyrolysis of the difluorocarbene source to provide difluorocarbene. For instance, the difluorocarbene source may be (triphenylphosponio)difluoroacetate and the difluorocarbene source, the 18F− source and the SO2 source may be heated to a temperature of from 80 to 150° C. The difluorocarbene source, the 18F− source and the SO2 source may be heated to a temperature of from 80 to 130° C., or a temperature of from 90 to 120° C., or a temperature of from 100 to 120° C.
The process of the present invention may further comprise a step of purifying the compound comprising the anion [CF218FSO2]−. Any suitable purification method may be employed; a wide range of suitable purification methods is well known to the skilled person. The step of purifying may comprise performing chromatography, for example weak anion exchange chromatography, high-performance liquid chromatography, reverse-phase high-performance liquid chromatography. The step of purifying may comprise performing several purification methods to obtain the compound comprising the anion [CF21SFSO2]−.
In one embodiment, the process of the present invention is a process for producing a compound comprising the anion [CF218FSO2]−, which process comprises treating (triphenylphosponio)difluoroacetate ([Ph3P]+CF2COO−) with (i) [18F]KF/K222 and (ii)N-methylmorpholine-SO2. Typically, the process is carried out at a temperature of from 80 to 150° C. Typically, the process is carried in out in a solution comprising a mixture of dimethyl formamide and propylene carbonate. Typically, this process produces CF218FSO2K. The process may comprise further purification steps that result in a final product that is CF218FSO2NH4.
Compound comprising [CF218FSO2]
The invention also provides a compound comprising the anion [CF218FSO2]−. Typically, the compound comprises a counter cation. Any suitable counter-cation may be employed; many such cations are known to the skilled person. Thus, the compound comprising the anion may be [CF218FSO2]−nAn+, wherein n is an integer of from 1 to 4. Preferably n is 1 or 2. In a preferred embodiment, n is 1.
Typically, the compound of the invention comprises at least 500 MBq of the anion [CF218FSO2]−. The compound of the invention may, for instance, comprise at least 600 MBq of the anion [CF218FSO2]−, or for instance at least 700 MBq of the anion, of rexample at least 800 MBq, at least 900 MBq, or at least 1 GBq of the anion.
The invention further provides a composition comprising the compound of the invention, wherein the composition comprises at least any of the above-mentioned amounts of the anion [CF218FSO2]−, in MBq or GBq.
In the compound of the invention, when n is 1, An+ is typically an alkali metal cation or an ammonium cation. An+ may be Li+, Na+, K+, Rb+, Cs+ or Fr+. Therefore the compound comprising the anion [CF28FSO2]− may be CF218FSO2Li, CF218FSO2Na, CF218FSO2K, CF218FSO2Rb, CF218FSO2Cs or CF218FSO2Fr. Often the compound is CF218FSO2K.
Ammonium cations include groups of formula [NRaRbRcRd]+, wherein Ra, Rb, Rc and Rd are each independently selected from H, substituted or unsubstituted C1-20 alkyl, substituted or unsubstituted C2-20 alkenyl, substituted or unsubstituted C2-20 alkynyl, substituted or unsubstituted C3-20 cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl; and wherein two or more of Ra, Rb, Rc and Rd may be bonded together to form one or more rings. Typically, Ra, Rb, Rc and Rd are each independently selected from hydrogen and unsubstituted C1-20 alkyl. Typically, the ammonium cation is [NH4]+, and the compound comprising the anion [CF218FSO2]− is CF218FSO2NH4.
When n is 2, An+ is typically a divalent metal cation. An+ may be a divalent metal cation selected from alkaline earth metal cations and divalent transition metal cations. For instance, An+ may be Mg2+, Ca2+, Sr2+, Ba2+, Sc2+, Ti2+, V2+, Cr2+, Mn2+, Fe2+, Ni2+, Cu2+ or Zn2+. An may for instance be Zn2+.
The compound comprising the anion [CF218FSO2] may be obtainable by the process of the invention as defined herein. The compound comprising the anion [CF218FSO2] may be obtained by the process of the invention as defined herein. For instance, the compound comprising the anion [CF218FSO2] may be obtained by a which process comprises treating a difluorocarbene source as defined anywhere herein with
Process for Functionalising Aromatic Groups
The present invention also provides a process for producing a compound comprising an 18F-trifluoromethyl functionalised aromatic group, which process comprises contacting a compound comprising an aromatic group with a compound comprising the anion [CF218FSO2]− in the presence of an activator for trifluoromethyl radical formation.
Typically the compound comprising an 18F-trifluoromethyl functionalised aromatic group is treated with the compound comprising the anion [CF218FSO2]− and the activator for trifluoromethyl radical formation simultaneously. For instance, the process may comprise contacting a mixture comprising the compound comprising an aromatic group and the compound comprising the anion [CF218FSO2]− with the activator for trifluoromethyl radical formation. The process may comprise contacting a mixture comprising the compound comprising an aromatic group and activator for trifluoromethyl radical formation with the compound comprising the anion [CF218FSO2]−. The process may comprise contacting a mixture comprising the activator for trifluoromethyl radical formation and the compound comprising the anion [CF218FSO2]− with the compound comprising an aromatic group.
An 18F-trifluoromethyl functionalised aromatic group corresponds to an aromatic group in which an 18F-trifluoromethyl group (—CF218F) has been bonded to an atom of the aromatic group. In the 18F-trifluoromethyl functionalised aromatic group the 18F-trifluoromethyl group replaces one of the substituents (either hydrogen or any other substituent as described herein, but typically hydrogen) on the aromatic group of the compound comprising an aromatic group.
Typically, the aromatic group is a substituted or unsubstituted aryl group or a substituted or unsubstituted heteroaryl group. When the aromatic group is a substituted or unsubstituted aryl group, the substituted or unsubstituted aryl group may be selected from substituted or unsubstituted phenyl, substituted or unsubstituted naphthyl, substituted or unsubstituted indenyl, substituted or unsubstituted indanyl, substituted or unsubstituted anthracenyl and substituted or unsubstituted pyrenyl. Typically the aromatic group is substituted or unsubstituted phenyl. Often, the aromatic group is an unsubstituted phenyl group or a phenol group.
When the aromatic group is a substituted or unsubstituted heteroaryl group, the substituted or unsubstituted heteroaryl group may be selected from substituted or unsubstituted pyridyl, substituted or unsubstituted pyrazinyl, substituted or unsubstituted pyrimidinyl, substituted or unsubstituted pyridazinyl, substituted or unsubstituted furanyl, substituted or unsubstituted thienyl, substituted or unsubstituted pyrazolidinyl, substituted or unsubstituted pyrrolyl, substituted or unsubstituted oxazolyl, substituted or unsubstituted oxadiazolyl, substituted or unsubstituted isoxazolyl, substituted or unsubstituted thiadiazolyl, substituted or unsubstituted thiazolyl, substituted or unsubstituted isothiazolyl, substituted or unsubstituted imidazolyl, substituted or unsubstituted pyrazolyl, substituted or unsubstituted triazolyl, substituted or unsubstituted indolyl, substituted or unsubstituted benzimidazolyl, substituted or unsubstituted indazolyl, substituted or unsubstituted benzotriazolyl, substituted or unsubstituted pyrrolopyridinyl, substituted or unsubstituted pyrrolopyrimidinyl, substituted or unsubstituted purinyl, substituted or unsubstituted indolizinyl, substituted or unsubstituted pyrrolopyrazinyl, substituted or unsubstituted pyrrolopyriminyl, substituted or unsubstituted pyrrolopyridazinyl, substituted or unsubstituted imidazopyridinyl, substituted or unsubstituted pyrazolopyridinyl, substituted or unsubstituted imidazopyridazinyl, substituted or unsubstituted imidazopyrimidinyl, substituted or unsubstituted imidazopyrazinyl, substituted or unsubstituted imidazopyrimidinyl, substituted or unsubstituted triazolopyridinyl, substituted or unsubstituted quinolyl, substituted or unsubstituted isoquinolyl, substituted or unsubstituted cinnolinyl, substituted or unsubstituted quinazolinyl, substituted or unsubstituted quinoxalinyl, substituted or unsubstituted phthalazinyl, substituted or unsubstituted pyridopyrazinyl, substituted or unsubstituted pteridinyl, substituted or unsubstituted pyridopyridazinyl, substituted or unsubstituted naphthyridinyl, and substituted or unsubstituted carbazolyl. The aromatic group may be a substituted or unsubstituted indole group or a substituted or unsubstituted imidazole group. For instance, the aromatic group may be an unsubstituted indolyl group or an unsubstituted imidazolyl group.
The compound comprising an aromatic group may comprise at least one aromatic group, at least two aromatic groups or at least three aromatic groups as described herein. The compound comprising an aromatic group may have a single aromatic group as described herein. The compound comprising an aromatic group may have a two aromatic groups as described herein. The compound comprising an aromatic group may have a three aromatic groups as described herein. The process may comprise functionalising at least one of the aromatic groups on the compound comprising an aromatic group, at least two of the aromatic groups on the compound comprising an aromatic group or at least three of the aromatic groups on the compound comprising an aromatic group. When the compound comprising an aromatic group comprises more than one aromatic group, 18F-trifluoromethylation may occur preferentially at one of the aromatic groups. When the compound comprising an aromatic group comprises more than one aromatic group, 18F-trifluoromethylation may occur at all of the aromatic groups present.
The compound comprising an aromatic group may be an amino acid or a compound comprising an amino acid. Preferably, the amino acid is tyrosine, tryptophan, phenylalanine or histidine. More preferably the amino acid is tyrosine or tryptophan. In some embodiments the amino acid is tyrosine. In some embodiments the amino acid is tryptophan. Hence, the compound comprising an aromatic group may be tyrosine, tryptophan, phenylalanine or histidine or a compound comprising tyrosine, tryptophan, phenylalanine or histidine. Preferably, the compound comprising an aromatic group is tyrosine or tryptophan or a compound comprising tyrosine or tryptophan.
The compound comprising an aromatic group may be a peptide or a protein.
When the compound comprising an aromatic group is a peptide, the peptide may be a dipeptide, a tripeptide, an oligopeptide or a polypeptide. The peptide may be an alkaloid, an anti-microbial agent, a hormone, a growth factor, an immunomodulating agent or an anti-oxidant.
The compound comprising an aromatic group may be a peptide or protein comprising tyrosine, tryptophan, phenylalanine or histidine. Preferably, the compound comprising an aromatic group is a peptide or protein comprising tyrosine or tryptophan. The compound comprising an aromatic group may be a peptide or protein comprising tyrosine and tryptophan.
The peptide or protein may comprise at least one of tyrosine, tryptophan, phenylalanine or histidine, at least two of tyrosine, tryptophan, phenylalanine or histidine or at least three of tyrosine, tryptophan, phenylalanine or histidine. In some instances, the peptide or protein comprises at least one tyrosine residue and at least one tryptophan residue, for example Endomorphin I. In some instances, the peptide or protein comprises at least one tyrosine residue and at least one phenylalanine residue, for example insulin. In some instances, the peptide or protein comprises at least one tyrosine residue and at least one histidine residue, for example Angiotensin I/II or insulin. In some instances, the peptide or protein comprises at least one tryptophan residue and at least one phenylalanine residue, for example Somatostatin-14. In some instances, the peptide or protein comprises at least one tryptophan residue and at least one histidine residue. In some instances, the peptide or protein comprises at least one phenylalanine residue and at least one histidine residue, for example insulin.
The peptide or protein may comprise at least one tyrosine residue, at least two tyrosine residues or at least three tyrosine residues. The peptide or protein may comprise at least one tryptophan residue, at least two tryptophan residues or at least three tryptophan residues. The peptide or protein may comprise at least one phenylalanine residue, at least two phenylalanine residues or at least three phenylalanine residues. The peptide or protein may comprise at least one histidine residue, at least two histidine residues or at least three histidine residues.
The compound comprising an aromatic group may be a peptide selected from Thymogen, Endomorphin I, Melittin, Angiotensin I/II, Insulin, Somatostatin-14 and cyclo(-Arg-Gly-Asp-D-Tyr-Lys):
Activators
The activator generates the 18F-trifluoromethyl radical from the compound comprising the anion [CF218FSO2]−. Any suitable activator known to the skilled person may be used. Typically, the activator comprises an oxidant, a photosensitizer, a photoredox catalyst or UV light. Examples of activators are discussed in the papers by Li et al. (Li., L et al. Simple and Clean Photoinduced Aromatic Trifluoromethylation Reaction, J. Am. Chem. Soc., 2016, 138, 5809-5812), Wang et al. (Wang., D. Catalyst-free direct C-H trifluoromethylation of arenes in water-acetonitrile, Green Chem., 2016, 18, 5967-5970) and Lefebvre (Lefebvre., Q, Toward Sustainable Trifluoromethylation Reactions: Sodium Triflinate under the Spotlight, Synlett 2017, 28, 19-23).
An oxidant is any substance capable of accepting electrons, thereby oxidising another compound present. Any suitable oxidant may be used. Examples of oxidants are well known to the skilled person. The oxidant may be an organic oxidant or an inorganic oxidant. For instance, the oxidant may be a compound comprising a peroxide group (—O—O—), a compound comprising I(III) or molecular oxygen (O2).
Examples of compounds comprising I(III) include, but are not limited to diacetoxyiodobenzene derivatives, e.g. phenyliodine bis(trifluoroacetate), and iodine pentoxide.
When the oxidant is a compound comprising a peroxide group, the compound comprising a peroxide group may be a compound of formula R—O—O—R, wherein each R is independently selected from hydrogen, substituted or unsubstituted C1-20 alkyl, substituted or unsubstituted C2-20 alkenyl, substituted or unsubstituted C2-20 alkynyl, substituted or unsubstituted C3-20 cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, substituted and unsubstituted heteroaryl and —SO3−. When both R groups are —SO3− the oxidant may be a persulfate salt, for example sodium persulfate (Na2S2O8).
Preferably, the oxidant is compound of formula R—O—O—R, wherein each R is independently selected from hydrogen and substituted or unsubstituted C1-10 alkyl. For instance, one R group may be hydrogen and the other R group may be selected from methyl, ethyl, propyl, n-butyl, s-butyl, i-butyl or t-butyl. Preferably the oxidant is t-butyl hydroperoxide (tBuOOH). Photosensitization is the process by which a photochemical or photophysical alteration occurs in one molecular entity as a result of initial absorption of radiation by another molecular entity called a photosensitizer. The photosensitizer may undergo a chemical change itself. Any suitable photosensitizer may be used. Examples of photosensitizers are well known to the skilled person. Typically when the activator comprises a photosensitizer, contacting the compound comprising an aromatic group with the compound comprising the anion [CF218FSO2]− in the presence of the photosensitizer is carried out in the presence of light. Typically the light is visible light or UV light.
The photosensitizer may be a compound of formula R[C(O)]nR wherein each R is independently selected from H, substituted or unsubstituted Ci-20 alkyl, substituted or unsubstituted C2-20 alkenyl, substituted or unsubstituted C2-20 alkynyl, substituted or unsubstituted C3-20 cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl and substituted and unsubstituted heteroaryl; wherein the two R groups, together with the —[C(O)]n— group to which they are attached may be bonded together to form a ring; and wherein n is an integer of from 1 to 5.
Typically, the photosensitizer is a compound of formula R[C(O)]nR wherein each R is independently substituted or unsubstituted C1-10 alkyl or substituted or unsubstituted aryl, and wherein n is 1 or 2. Preferably, the photosensitizer is a compound of formula R[C(O)]nR wherein R is substituted or unsubstituted C1-6 alkyl and wherein n is 1 or 2. Preferably, the photosensitizer is acetone, diacetyl or a combination thereof.
A photoredox catalyst is a compound that, when excited by light, can mediate the transfer of electrons. Any suitable photoredox catalyst may be used. Examples of photoredox catalysts are well known to the skilled person. The photoredox catalyst may be an organic photoredox catalyst or an inorganic photoredox catalyst. Typically when the activator comprises a photoredox catalyst, contacting the compound comprising an aromatic group with a compound comprising the anion [CF218FSO2]− in the presence of the photoredox catalyst is carried out in the presence of light. Typically the light is visible light or UV light.
Examples of photoredox catalysts include, but are not limited to transition metal complexes, such as Ru or Ir complexes, or conjugated organic compounds, such as benzophenone derivatives, anthraquinone derivatives and acridinium derivatives. The photoredox catalyst may be Ru(bipy)3, Ir[dF(CF3)ppy]2(dtbbpy)PF6, N-Me-mesitylacridinium, anthraquinone-2-carboxylic acid or dimethoxybenzophenone.
The activator may comprise UV light. In one embodiment, the activator is UV light, for instance UV light with a wavelength of less than 280 nm. The activator may comprise UV light and either a photosensitizer or a photoredox catalyst as described herein.
Additive
The step of contacting the compound comprising an aromatic group with a compound comprising the anion [CF218FSO2]− in the presence of an activator for trifluoromethyl radical formation may be performed in the presence of an additive. The additive may be any compound that influences the rate of the reaction or the product distribution of the reaction.
The additive may be a metal salt, for example a transition metal salt. The additive may be the salt of a first row transition metal, for example a salt of Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu or Zn, preferably a salt of Fe. The additive may be a transition metal (III) salt. Preferably, the additive is an Fe(III) salt.
When the additive is a metal salt, the additive will typically comprise an anion. The anion may be selected from any anion described herein. For instance, the one or more anions may be selected from halide, hydroxide, sulfate, and nitrate. Typically the anion is chloride or nitrate. The anion is often nitrate. Thus, the additive may be a metal nitrate salt, typically a transition metal nitrate salt, for instance a first row transition metal nitrate salt, for example iron nitrate. Typically, the additive is Fe(III) nitrate. The metal salt may be a hydrate. For instance, the additive may be Fe(NO3)3.9H2O. The anion is often chloride. The additive may be a metal chloride salt, typically a transition metal chloride salt, for instance a first row transition metal chloride salt, for example iron chloride. Typically the additive is Fe(III) chloride. The metal salt may be a hydrate. For instance, the additive may be FeCl3.6H2O.
In one embodiment, the activator is an oxidant as described herein and the additive is a metal salt as described herein. Typically, the activator is a compound comprising a peroxide (—O—O—) group and the additive is a transition metal salt. Preferably, the activator is t-butyl hydroperoxide and the additive is Fe(III) nitrate, preferably Fe(NO3)3.9H2O, or Fe(III) chloride, preferably FeCl3.6H2O. The addition of a transition metal salt, such as Fe(NO3)3.9H2O or FeCl3.6H2O permits the fast addition of t-butyl hydroperoxide to the reaction mix without oxidation of the [CF218FSO2]− anion to [CF218FSO3]−.
In the process for producing a compound comprising an 18F-trifluoromethyl functionalised aromatic group of the invention, the compound comprising the anion [CF218FSO2]− may be as described herein.
The compound comprising the anion [CF218FSO2]− may be obtainable by a process as defined herein. The process for producing a compound comprising an 18F-trifluoromethyl functionalised aromatic group may further comprise a step of obtaining the compound comprising the anion [CF218FSO2]− by a process as defined herein.
In one embodiment, the process for producing a compound comprising an 18F-trifluoromethyl functionalised aromatic group comprises contacting a compound comprising an aromatic group with CF218FSO2NH4 in the presence of t-butyl hydroperoxide and Fe(NO3)3.9H2O.
Compound Comprising an 18F-Trifluoromethyl Functionalised Aromatic Group
The present invention also provides a compound comprising an 18F-trifluoromethyl functionalised aromatic group. The aromatic group may be any aromatic group as described herein.
The compound comprising an 18F-trifluoromethyl functionalised aromatic group may comprise at least one aromatic group, at least two aromatic groups or at least three aromatic groups as described herein. The compound comprising an 18F-trifluoromethyl functionalised aromatic group may have a single aromatic group as described herein. The compound comprising an aromatic group may have two aromatic groups as described herein. The compound comprising an aromatic group may have three aromatic groups as described herein.
When the compound comprising a 18F-trifluoromethyl functionalised aromatic group contains multiple aromatic groups, it is not necessary that all of the aromatic groups are 18F-trifluoromethyl functionalised. When the compound comprising an aromatic group comprises more than one aromatic group, one of the aromatic groups may be an 18F-trifluoromethyl functionalised aromatic group. When the compound comprising an aromatic group comprises more than one aromatic group, all of the aromatic groups may be 18F-trifluoromethyl functionalised aromatic groups. The compound comprising an 18F-trifluoromethyl functionalised aromatic group may comprise at least one 18F-trifluoromethyl functionalised aromatic group, at least two 18F-trifluoromethyl functionalised aromatic groups or at least three 18F-trifluoromethyl functionalised aromatic groups.
In one embodiment, the compound comprising an 18F-trifluoromethyl functionalised aromatic group is an amino acid or a compound comprising an amino acid. Thus, the compound may be an 18F-trifluoromethyl functionalised amino acid, or a compound comprising an 18F-trifluoromethyl functionalised amino acid. Typically, the amino acid is tyrosine, tryptophan, phenylalanine or histidine. Often the amino acid is tyrosine or tryptophan. In some embodiments the amino acid is tyrosine. In some embodiments the amino acid is tryptophan.
Thus, the compound comprising an 18F-trifluoromethyl functionalised aromatic group may be 18F-trifluoromethyl functionalised tyrosine, 18F-trifluoromethyl functionalised tryptophan, 18F-trifluoromethyl functionalised phenylalanine or 18F-trifluoromethyl functionalised histidine. The compound comprising an 18F-trifluoromethyl functionalised aromatic group may be a compound comprising 18F-trifluoromethyl functionalised tyrosine, 18F-trifluoromethyl functionalised tryptophan, 18F-trifluoromethyl functionalised phenylalanine or 18F-trifluoromethyl functionalised histidine. Preferably, the compound comprising an 18F-trifluoromethyl functionalised aromatic group is 18F-trifluoromethyl functionalised tyrosine or 18F-trifluoromethyl functionalised tryptophan or a compound comprising 18F-trifluoromethyl functionalised tyrosine or 18F-trifluoromethyl functionalised tryptophan.
The compound comprising an 18F-trifluoromethyl functionalised aromatic group may be a peptide or a protein. Thus, the compound may be an 18F-trifluoromethyl functionalised peptide or an 18F-trifluoromethyl functionalised protein.
When the compound comprising an 18F-trifluoromethyl functionalised aromatic group is a peptide, the peptide may be a dipeptide, a tripeptide, an oligopeptide or a polypeptide. The peptide may be an alkaloid, an anti-microbial agent, a hormone, a growth factor, an immunomodulating agent or an anti-oxidant.
The compound comprising an 18F-trifluoromethyl functionalised aromatic group may be a peptide or protein comprising 18F-trifluoromethyl functionalised tyrosine, 18F-trifluoromethyl functionalised tryptophan, 18F-trifluoromethyl functionalised phenylalanine or 18F-trifluoromethyl functionalised histidine. Preferably, the compound comprising an aromatic group is a peptide or protein comprising 18F-trifluoromethyl functionalised tyrosine or 18F-trifluoromethyl functionalised tryptophan. The compound comprising an aromatic group may be a peptide or protein comprising 18F-trifluoromethyl functionalised tyrosine and 18F-trifluoromethyl functionalised tryptophan.
The compound comprising an 18F-trifluoromethyl functionalised aromatic group may be a peptide or protein comprising at least one of tyrosine, tryptophan, phenylalanine or histidine, at least two of tyrosine, tryptophan, phenylalanine or histidine or at least three of tyrosine, tryptophan, phenylalanine or histidine. In some instances, the compound comprising a 18F-trifluoromethyl functionalised aromatic group is a peptide or protein comprising at least one tyrosine residue and at least one tryptophan residue, for example Endomorphin I. In some instances, the compound comprising a 18F-trifluoromethyl functionalised aromatic group is a peptide or protein comprising at least one tyrosine residue and at least one phenylalanine residue, for example insulin. In some instances, the compound comprising a 18F-trifluoromethyl functionalised aromatic group is a peptide or protein comprising at least one tyrosine residue and at least one histidine residue, for example Angiotensin I/II or insulin. In some instances, the compound comprising a 18F-trifluoromethyl functionalised aromatic group is a peptide or protein comprising at least one tryptophan residue and at least one phenylalanine residue, for example Somatostatin-14. In some instances, the compound comprising a 18F-trifluoromethyl functionalised aromatic group is a peptide or protein comprising at least one tryptophan residue and at least one histidine residue. In some instances, the compound comprising a 18F-trifluoromethyl functionalised aromatic group is a peptide or protein comprising at least one phenylalanine residue and at least one histidine residue, for example insulin.
The compound comprising a 18F-trifluoromethyl functionalised aromatic group may be a peptide or protein comprising at least one tyrosine residue, at least two tyrosine residues or at least three tyrosine residues. The compound comprising a 18F-trifluoromethyl functionalised aromatic group may be a peptide or protein comprising at least one tryptophan residue, at least two tryptophan residues or at least three tryptophan residues. The compound comprising a 18F-trifluoromethyl functionalised aromatic group may be a peptide or protein comprising at least one phenylalanine residue, at least two phenylalanine residues or at least three phenylalanine residues. The compound comprising a 18F-trifluoromethyl functionalised aromatic group may be a peptide or protein comprising at least one histidine residue, at least two histidine residues or at least three histidine residues.
The compound comprising an 18F-trifluoromethyl functionalised aromatic group may be a peptide selected from Thymogen, Endomorphin I, Melittin, Angiotensin I/II, Insulin, Somatostatin-14 and cyclo(-Arg-Gly-Asp-D-Tyr-Lys). For instance, the compound may be Thymogen in which tryptophan is functionalised with a 18F-trifluoromethyl group, Endomorphin I in which tryptophan is functionalised with a 18F-trifluoromethyl group, Melittin in which tryptophan is functionalised with a 18F-trifluoromethyl group, Angiotensin I/II in which tyrosine is functionalised with a 18F-trifluoromethyl group, insulin in which tyrosine is functionalised with a 18F-trifluoromethyl group, somatostatin-14 in which tryptophan is functionalised with a 18F-trifluoromethyl group or cyclo(-Arg-Gly-Asp-D-Tyr-Lys) in which tyrosine is functionalised with a 18F-trifluoromethyl group.
Thus, the compound comprising 18F-trifluoromethyl functionalised aromatic group may be selected from:
The compound comprising 18F-trifluoromethyl functionalised aromatic group may be obtainable by a process of the invention as defined herein. The compound comprising 18F-trifluoromethyl functionalised aromatic group may be obtained by a process of the invention as defined herein.
The invention also provides a pharmaceutical composition comprising (i) a compound comprising an 18F-trifluoromethyl functionalised aromatic group, or a pharmaceutically acceptable salt thereof, and optionally (ii) one or more pharmaceutically acceptable ingredients.
Suitable pharmaceutically acceptable ingredients are well known to those skilled in the art and include pharmaceutically acceptable carriers (e.g. a saline solution, an isotonic solution), diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers, solubilisers, surfactants (e.g. wetting agents), masking agents, colouring agents, flavouring agents and sweetening agents. Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical texts. See, for example, Handbook for Pharmaceutical Additives, 2nd Edition (eds. M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, N.Y., USA), Remington's Pharmaceutical Sciences, 20th edition, pub. Lippincott, Williams & Wilkins, 2000; and Handbook of Pharmaceutical Excipients, 2nd edition, 1994.
A pharmaceutical composition may be in the form of (i.e. be formulated as) a liquid, a solution or a suspension (e.g. an aqueous or a non-aqueous solution), an emulsion (e.g. oil-in-water, water-in-oil), an elixir, a syrup, an electuary, a tablet (e.g. coated tablets), granules, a powder, a lozenge, a pastille, a capsule (e.g. hard and soft gelatine capsules), a pill, an ampoule, a bolus, a tincture, a gel, a paste or an oil.
Typically the pharmaceutical composition is suitable for parenteral administration. A pharmaceutical composition suitable for parenteral administration (e.g. by injection) may include an aqueous or non-aqueous, sterile liquid in which the particles employed in the invention are dissolved or suspended. Such liquids may additionally contain other pharmaceutically acceptable ingredients, such as anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, suspending agents, thickening agents, and solutes that render the formulation isotonic with the blood (or other relevant bodily fluid) of the intended recipient. Examples of excipients include water, alcohols, polyols, glycerol, vegetable oils, and the like. Examples of suitable isotonic solutions for use in such formulations include Sodium Chloride Injection, Ringer's Solution or Lactated Ringer's Injection.
The invention also provides a compound comprising an 18F-trifluoromethyl functionalised aromatic group as described herein for use in a method for treatment of the human or animal body by therapy or for use in a diagnostic method practised on the human or animal body.
The invention also provides a method of treatment comprising administering a therapeutically effective amount of a compound comprising an 18F-trifluoromethyl functionalised aromatic group as described herein to a subject.
The invention also provides the use of a compound comprising an 18F-trifluoromethyl functionalised aromatic group as described herein in the manufacture of a medicament.
The invention also provides a method of imaging a subject, comprising administering to the subject a compound comprising 18F-trifluoromethyl functionalised aromatic group as described herein or a pharmaceutically acceptable salt thereof, and imaging the subject by positron emission tomography (PET). The method may comprise administering to the subject a pharmaceutical composition as described herein.
The invention is further described in the following Examples.
For radiochemistry, the approaches in scheme 1A above are convoluted because they would require a radiosynthetic route towards the necessary [18F]CF3-precursor, and one or more reactions post-labeling. Our design plan was to construct [18F]CF3SO2-applying a multi-component approach that would combine 18F-fluoride, a difluorocarbene source, and SO2 one-pot. The formation of the 18F-trifluoromethyl anion from difluorocarbene and 18F-fluoride is known (Huiban, M et al., Nat. Chem. 2013, 5 (11), 941-944; Zheng, J. et a., Angew. Chem., Int. Ed. 2015, 54 (45), 13236-13240; Zheng, J. et al., Angew. Chem., Int. Ed. 2017, 56 (12), 3196-3200.). A challenge associated with our proposed approach was to validate a protocol that couples the in situ generated [18F]CF3− with SO2, as illustrated in Scheme 1B, preferably but not necessarily using an SO2 source other than SO2 itself (so that this gaseous toxic reagent is not needed).
Exploratory studies performed with 19F-fluoride provided useful information (see below). Both the difluorocarbene and SO2 sources were found critical to enable the construction of CF3SO2−. The reaction of (triphenylphosphonio)-difluoroacetate (PDFA) with either 1,4-diazabicyclo[2.2.2]-octane bis(SO2) adduct (DABSO)30 or N-methyl-morpholine.SO2 (NMM.SO2) in the presence of KF/K222 in DMF at 120° C. afforded CF3SO2K in 63% and 52% yield (19F NMR), respectively. ClF2CCO2Me in combination with PPh3 was found to be suitable for this process. In contrast to experiments carried out with 19F-fluoride, DABSO afforded [18F]CF3SO2K in only trace amount (Scheme 2A). The combination of PDFA, NMM.SO2 and [18F]KF/K222 (typically 20-30 MBq) gave [18F]CF3SO2K in 16% RCC. These results encouraged the development of a protocol to prepare, purify and isolate this novel 18F-reagent for subsequent use (Scheme 2B). PDFA is thermally unstable and poorly soluble in DMF, so these limitations require that a mixture of this reagent and NMM.SO2, was added as a suspension in DMF to a vial containing azeotropically dried 18F-fluoride. Amongst all solvents tested, propylene carbonate (PC) was the most suitable when used with DMF (see See: Yang, Y.; Xu, L.; Yu, S.; Liu, X.; Zhang, Y.; Vicic, D. A. Chem.—A Eur. J. 2016, 22 (3), 858-863). Additional optimization tuning the ratio of reagents and concentration proved beneficial. The optimal process consisted of reacting PDFA (0.16 mmol) and NMM.SO2 (0.06 mmol) with [18F]KF/K222 (up to 10 GBq) in 350 μL PC/DMF. Initial purification of [18F]CF3SO2K using a weak anion exchange cartridge (WAX) allowed for most of the unreacted 18F-fluoride and organic byproducts to be removed. Elution with a solution of ˜0.4 M ammonia in EtOH followed by reverse phase HPLC purification afforded [18F]CF3SO2NH4 in >99% radiochemical purity. Using this protocol, up to 900 MBq of [18F]CF3SO2NH4 could be isolated from 10 GBq of 18F-fluoride. The overall nondecay corrected activity yield of isolated [18F]1 calculated from 18F-fluoride is 10%±1% (n=7). The identity of [18F]CF3SO2NH4 was established by HPLC and mass spectrometry ([19F]CF3SO2− (m/z 133.1, calcd 133.0).
18F-fluoride
aPC = propylene carbonate. b300 μL. c350 μL. dRadiochemical purity (RCP) > 99%
Studies towards C—H 18F-trifluoromethylation began using model peptides containing tyrosine and/or tryptophan using t-butyl hydroperoxide (TBHP) as the oxidant. We were faced with immediate challenges. In 19F-mode, CF3SO2Na is added in large excess to enable C—H trifluoromethylation of peptides and proteins (up to ˜200 equiv) (see Imiolek, M.; Karunanithy, G.; Ng, W. L.; Baldwin, A. J.; Gouvemeur, V.; Davis, B. G. J. Am. Chem. Soc. 2018, 140 (5), 1568-1571 and Ji, Y.; Brueckl, T.; Baxter, R. D.; Fujiwara, Y.; Seiple, I. B.; Su, S.; Blackmond, D. G.; Baran, P. S. Proc. Natl. Acad. Sci. 2011, 108 (35), 14411-14415). These conditions are not compatible with 18F-radiochemisty due to inherent constraints on concentration pertaining to large peptide or proteins, and the 18F-reagent. An additional complication is competitive oxidation of [19F]CF3SO2NH4 into [18F]CF3SO3NH4 in the presence of the initiation reagent. For 19F-trifluoromethylation, this issue is solved either by using an excess of [19F]CF3SO2Na with respect to TBHP, or via slow addition of TBHP to the reaction mixture (see Ji, Y.; Brueckl, T.; Baxter, R. D.; Fujiwara, Y.; Seiple, I. B.; Su, S.; Blackmond, D. G.; Baran, P. S. Proc. Natl. Acad. Sci. 2011, 108 (35), 14411-14415). These solutions are not suitable for 18F-labeling because [18F]CF3SO2NH4 will be the limiting reagent, and operational simplicity is paramount for 18F-radiochemistry.
The treatment of L-Tyr (0.12 mmol) with [18F]CF3SO2NH4 (8 MBq) and TBHP (0.12 mmol) in aqueous NH4HCO2 (AF) buffer (10% AcOH) did not lead to C—H 18F-trifluoromethylation at 60° C. after 20 mins. Extensive optimization of the reaction parameters led to [18F]o-CF3-L-Tyr in 18% RCC in the presence of both TBHP and Fe(NO3)3.9H2O (Scheme 3A). Higher RCC of 50% was obtained at 60° C. Trifluoromethylation occurred mainly on the o-position, but m-substituted 18F—CF3-product was also detected (3% RCC). These two isomers are separable by HPLC. The C—H [18F]trifluoromethylation of L-Trp was also successful with [18F]CF3SO2NH4 activated by TBHP and FeC3. This reaction best performed in DMSO/AF afforded [18F]CF3-L-Trp in 27% RCC. The major product is [18F]2-CF3-L-Trp (17% RCC) but careful analysis indicated that two regioisomers resulting from competitive 18F-labeling at the 4- and 7-position were formed (the combined RCC for these two isomers is 10%). The treatment of L-Tyr (0.12 mmol) with [18F]CF3SO2NH4 (8 MBq) and TBHP (0.12 mmol) in aqueous NH4HCO2 (AF) buffer (10% AcOH) did not lead to C—H 18F-trifluoromethylation at 60° C. after 20 mins. Extensive optimization of the reaction parameters led to [18F]o-CF3-L-Tyr in 18% RCC in the presence of both TBHP and Fe(NO3)3.9H2O (Scheme 3A). Higher RCC of 50% was obtained at 60° C. Trifluoromethylation occurred mainly on the o-position, but m-substituted 18F—CF3-product was also detected (3% RCC). These two isomers are separable by HPLC. The C—H [18F]trifluoromethylation of L-Trp was also successful with [18F]CF3SO2NH4 activated by TBHP and FeCl3. This reaction best performed in DMSO/AF afforded [18F]CF3-L-Trp in 27% RCC. The major product is [18F]2-CF3-L-Trp (17% RCC) but careful analysis indicated that two regioisomers resulting from competitive 18F-labeling at the 4- and 7-position were formed (the combined RCC for these two isomers is 10%).
Next, a series of dipeptides was evaluated with a focus on feasibility and selectivity (Scheme 3B). For reactions leading to more than one 18F-labeled product, identification was made by comparison of HPLC traces with authentic references prepared independently and fully characterized. Dipeptides Tyr-Trp (Y-W), Trp-Tyr (W-Y) underwent 18F—CF3 incorporation exclusively at Trp with higher RCC obtained for the former. For dipeptide Phe-Tyr (F-Y), 18F-trifluoromethylation occurs at Y affording F-o[1BF]CF3Y in 37% RCC. Analysis of the crude reaction mixture indicated the formation of minor isomers resulting from [1′F]CF3-ation on Y at the meta position and at F. This competitive process was not observed for F-W[18F]CF3, a result consistent with the higher reactivity of W versus Y. No competitive 18F-labeling was detected at His (H) for both H-W[18F]CF3 and Y[18F]CF3—H. Met (M) oxidation was not observed when 18F-labeling of M-W (37% RCC), and was largely minimized for M-Y by increasing the Fe:TBHP ratio to 1:1. Oxidative dimerization of cysteine residue however is unavoidable. Next, we studied the 18F-labeling of biologically relevant peptides of increasing complexity. The dipeptide immunomodulator Thymogen or oglufanide was successfully 18F-trifluoromethylated at W, and isolated in 37% RCY. Endomorphin 1, a tetrapeptide associated with Alzheimer disease, also underwent W-selective 18F-labeling in 17% RCY. Similarly, Somatostatin-14, a cyclic tetradecapeptidic hormone with broad inhibitory effect on endocrine secretion, was 18F-labeled in 20% RCY. The 18F-trifluoromethylation of the larger 26-residues antimicrobial peptide Melittin was equally successful (18% RCC). Tyrosine-containing peptides were examined next. Angiotensin(1-7), a peptide which shows activity against human lung cancer cells, underwent 18F-labeling at Tyr in 9%±2% RCC. At this stage, the C—H 18F-trifluoromethylation of a much larger peptide was considered with recombinant human insulin (MW: about 5800 Da). This experiment carried out with 5.2 μmol of insulin, Fe(NO3)3.9H2O (5.8 equiv) and TBHP (11.5 equiv) in DMSO/25 mM aq. AF (150 μL) led to 18F—CF3-insulin as a mixture of four products resulting from [18F]CF3 incorporation at all tyrosine residues in 21% overall RCC. The main site of 18F-trifluoromethylation is chain A Y19, a result consistent with the report of Krsha et al. (see Ichiishi, N.; Caldwell, J. P.; Lin, M.; Zhong, W.; Zhu, X.; Streckfuss, E. C.; Kim, H.-Y. Y.; Parish, C. A.; Krska, S. W. Chem. Sci. 2018, 9 (17), 4168-4175). This is the largest unmodified peptide 18F-labeled to date.
In conclusion, we have developed the first protocol enabling direct 18F-labeling of unmodified peptides at the tryptophan and tyrosine residues (with high selectivity for tryptophan) with the CF3 group via innate C—H functionalization. This convenient method based on the use of readily available 18F-fluoride is a new tool to accelerate the discovery of 18F-peptides as imaging agents as well as the development of peptide-based drugs. The strategy required the designed 18F-isotopologue of the trifluoromethylsulfenate anion [18F]CF3SO2). Considering the number of reactions relying on the Langlois and Baran reagents, we anticipate that the availability of [18F]CF3SO2NH4 will expand considerably the radiochemical space for PET applications well beyond the peptides defined herein.
General Experimental Information
All NMR spectra were recorded on Bruker AVIII HD 400, AVII 500 and AVIII HD 500 spectrometers. Proton and carbon-13 NMR spectra are reported as chemical shifts (6) in parts per million (ppm) relative to the solvent peak values as given in Gottlieb et al. (Gottlieb, H. E.; Kotlyar, V.; Nudelman, A. J. Org. Chem. 1997, 62 (21), 7512-7515). Fluorine-19 NMR spectra are referenced relative to CFCl3 in CDCl3. If trifluoroacetate peak is present, it is used as a reference with values in the relevant solvent as given in Rosenau et al. (Rosenau, C. P.; Jelier, B. J.; Gossert, A. D.; Togni, A.; Rosenau, C. P.; Jelier, B. J.; Gossert, A. D. Angew. Chem., Int. Ed. 2018). Coupling constants (J) are reported in units of hertz (Hz). The following abbreviations are used to describe multiplicities—s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br. s (broad singlet). High resolution mass spectra (HRMS, m/z) were recorded on a Bruker MicroTOF spectrometer using positive electrospray ionization (ESI+). Infrared spectra were recorded either as the neat compound or in a solution using a Bruker Tensor 27 FT-IR spectrometer. Absorptions are reported in wavenumbers (cm1). Optical rotations were measured on a PerkinElmer Polarimeter model 341. Specific rotations are reported in concentrations in g/100 mL. Melting points of solids were measured on a Griffin apparatus and are uncorrected. IUPAC names were obtained using Perkin Elmer Chemdraw Professional Version 16.0.14(77). Solvents were purchased from Sigma-Aldrich, Honeywell and Fisher. Chemicals were purchased from Acros, Alfa Aesar, Bachem, Fluorochem, Sigma-Aldrich and used as received. Peptides were purchased from Bachem and used as received.
The LC-MS/MS analyses were recorded on Xevo G2 Q-TOF coupled to ACQUITY UPLC H-Class LC system (Waters Corporation). ESI ionization source parameters: capillary 3 kV, cone voltage 40 V, source temperature 100° C., desolvation temperature 400° C. and desolvation gas (nitrogen) flow 700 L/h. MS was working at 30,000 (FWHM) resolution. The mass spectrometer operated in data dependent acquisition (DDA) mode with MS survey scan (0.1 s) followed by 0.1 s-0.5 s MS/MS scans on the three most intense ions. To avoid optimization of collision conditions broad MS/MS Collision Energy Ramp 15 V-60 V was used. Leucine enkephalin was used as the lock mass standard. MS/MS data was processed using PEAKS Studio software v8.0 (Bioinformatics Solutions Inc.) with DeNovo analysis with following parameters parent mass error tolerance 10.0 ppm; fragment mass error tolerance 0.02 Da; monoisotopic precursor mass search type. Additional identification in selected cases was accomplished with spectral interpretation software (High Chem Mass Frontier 7.0). Peptide samples (10-50 μg/mL) were prepared in (10-50%) ACN:H2O mixture with 0.1% FA or TFA. Disulphide containing peptides (insulin, somatostatin) were reduced before LC-MS/MS analysis by mixing with a solution of 1M DTT in 100 mM NH4OAc buffer (pH 9.0) and 1 h room temperature incubation. The final concentration of DTT was 2 mM and basic pH was confirmed with pH indicator strips. Melittin (0.4 μg) sample was digested at 37° C. with porcine trypsin in 50 mM PBS at pH 8.0 overnight with protein to enzyme ratio 1:25. The reduction/digestion was terminated by acidification with 0.2% FA.
Synthesis Procedure
To a solution of potassium hydroxide (5.61 g, 100 mmol) in methanol (90 mL) was added ethyl bromodifluoroacetate (12.8 mL, 100 mmol) before stirring at rt for 12 h. The solvent was evaporated on a rotary evaporator and dried in vacuo to obtain a white solid (19.4 g, 91.1 mmol, 91%). Characterization data is consistent with those reported in the literature.3 19F NMR (377 MHz, Methanol-d4): δ −58.05 (s). 13C NMR (101 MHz, Methanol-d4) δ 164.98 (t, J=24.9 Hz), 115.22 (t, J=320.4 Hz).
A solution of potassium 2-bromo-2,2-difluoroacetate (2.13 g, 10.0 mmol) and triphenylphosphine (2.62 g, 10.0 mmol) in dry DMF (10 mL) was stirred at rt for 16 h. The mixture was filtered, and the residue was washed with copious amounts of water, acetone and diethyl ether before drying in vacuo to obtain a white solid (2.68 g, 7.53 mmol, 75%). Characterization data is consistent with those reported in the literature.3 1H NMR (400 MHz, Methanol-d4): δ 7.73-7.81 (m, 2H), 7.82-7.98 (m, 3H); 19F NMR (377 MHz, Methanol-d4): δ −96.04 (d, J=96.4 Hz); 31P NMR (162 MHz, Methanol-d4): δ 27.13 (t, J=96.8 Hz).
To a dry three neck round-bottom flask containing a magnetic stir bar, with a dry ice/acetone condenser, nitrogen inlet and bubbler attached, was added 4-methylmorpholine (5.94 mL, 50.0 mmol) before cooling with a dry ice/MeCN bath. SO2 gas was added until approximately 100 mL had condensed. The dry ice/MeCN bath was replaced with a water bath and the excess SO2 was evaporated while stirring under a nitrogen stream. The resultant solid was dried overnight over P2O5 to obtain a pale-yellow solid (7.38 g, 44.7 mmol, 89%). The solid was stored at −20° C. under an inert atmosphere as it is hygroscopic and slowly decomposes in air. 1H NMR (500 MHz, Methanol-d4) δ 3.94 (t, J=5.0 Hz, 4H), 3.28 (s, 4H), 2.88 (s, 3H). 13C NMR (101 MHz, Methanol-d4) δ 65.03, 54.47, 44.06, 44.05. 15N NMR (51 MHz, Methanol-d4) δ 40.62. Indirect observation from HMBC. IR (solid): 615.0, 623.8, 644.5, 765.2, 859.8, 893.4, 900.5, 936.8, 996.2, 1028.9, 1044, 1067.2, 1088.2, 1112.9, 1156.5, 1181.6, 1198.3, 1280.4, 1313.4, 1370.6, 1460.0, 2866.2, 2966.5, 3026.1. M.P.: 40° C. Analysis calculated for C5H11NO3S: C, 36.35; H, 6.71; N, 8.48; 0, 29.05; S, 19.41 found C, 35.32; H, 6.88; N, 8.14; 0, 31.03; S, 18.64.
General Trifluoromethylation Procedure 1
To a mixture of substrate (0.1 mmol), sodium triflinate (15.6 mg, 0.1 mmol), and iron (III) nitrate nonahydrate (40.4 mg, 0.1 mmol) in 1:9 acetic acid/25 mM aqueous ammonium formate (1.0 mL) was added tert-butyl hydroperoxide solution (27.6 μL, 0.2 mmol, 70% in water) before stirring at 40° C. for 20 mins. The solution was taken up in water (10 mL) and added to a Waters Oasis HLB cartridge (activated with 2 mL methanol, 10 mL water) before eluting the crude product with methanol (5.0 mL). The methanol was evaporated, and the desired product was purified via reverse phase preparative HPLC and the collected fractions were lyophilized.
General Trifluoromethylation Procedure 2
To substrate (0.02 mmol) in a 3 mL screw top V-Vials® with open-top cap (Sigma-Aldrich Z115142) was added 100 μL of DMSO, iron (III) nitrate nonahydrate (0.04 mmol, 40 μL of a 1M stock solution in 25 mM aqueous NH4HCO2), NaSO2CF3 (0.030 mmol, 30 μL of a 1M stock solution in 25 mM aqueous NH4HCO2), and 70% TBHP in water (as specified). The vial was placed in a water bath at 40° C. and stirred at 750 rpm for 20-60 mins.
L-Tyr[ortho-CF3]
Synthesised following the general trifluoromethylation procedure 1 to obtain a white solid. 1H NMR (400 MHz, Methanol-d4) δ 7.46 (d, J=2.2 Hz, 1H), 7.35 (dd, J=8.4, 2.2 Hz, 1H), 6.93 (d, J=8.3 Hz, 1H), 3.76 (dd, J=8.2, 4.6 Hz, 1H), 3.23 (dd, J=14.7, 4.5 Hz, 1H), 3.00 (dd, J=14.7, 8.2 Hz, 1H). Phenolic OH, Carboxylic OH and amine NH2 were not observed. 13C NMR (101 MHz, Methanol-d4) δ 173.52, 156.56, 135.33, 128.78 (q, J=5.1 Hz), 127.45, 126.52 (q, J=271.1 Hz), 118.30, 118.07 (q, J=30.8 Hz), 57.39, 37.12. 19F NMR (376 MHz, Methanol-d4) δ −61.45. HRMS (ESI+): for C10H11F3NO3 [M+H]+ requires m/z=250.0686, found 250.0684.
L-Tyr[meta-CF3]
Synthesised following the general trifluoromethylation procedure 1 to obtain a white solid. 1H NMR (500 MHz, DMSO-d6) δ 7.33 (d, J=8.5 Hz, 1H), 7.02 (d, J=2.7 Hz, 1H), 6.94 (dd, J=8.3, 2.6 Hz, 1H). Observed indirectly from COSY as these were obscured by the solvent peak: 3.30, 3.26, 2.71. 13C NMR (126 MHz, DMSO-d6) δ 133.15, 112.24, 118.75, 55.37, 33.72. Observed indirectly from HSQC. 19F NMR (470 MHz, DMSO-d6) δ −58.33. HRMS (ESI+): for C10H11F3NO3 [M+H]+ requires m/z=250.0686, found 250.0686.
L-Trp[W-2-CF3]
Synthesized following the general trifluoromethylation procedure 2. White solid was obtained after lyophilization. Characterization data is consistent with literature.4 Reverse phase HPLC details: Phenomenex Synergi™ 4 μm Hydro RP 80 Å, 250×10 mm. Flow rate=4 mL/min. Column temperature=40° C. Eluent=20% MeCN (0.1% TFA) and 80 H2O (0.1% TFA) for 3 mins then increase linearly to 27% MeCN at 15 mins and hold at 27% MeCN for another 3 mins. Retention time=9.4 min. 1H NMR (500 MHz, DMSO-d6) δ 12.12 (s, 1H), 8.27 (s, 3H), 7.76 (d, J=8.1 Hz, 1H), 7.46 (d, J=8.2 Hz, 1H), 7.31 (t, J=7.6 Hz, 1H), 7.16 (t, J=7.5 Hz, 1H), 3.87 (s, 1H), 3.43-3.27 (m, 2H) partially obscured by water peak. Carboxylic acid OH was not observed. 19F NMR (471 MHz, DMSO-d6) δ −57.81, −74.95. MS (ESI+): for C12H12F3N2O2 [M+H]+ requires 273.1 found 273.1.
Major Trifluoromethylated Side Products of L-Trp Reaction
W-4-CF3 data
Assignment of position on the indole is based on chemical shift of H for the unmodified tryptophan. 4—is more de-shielded than 7—therefore the 19F of the CF3 at 4—is expected to be less negative (more positive) than 7-. This assignment is also support by NOESY. Retention time=16.2 mins. 1H NMR (500 MHz, DMSO-d6) δ 11.44 (s, 1H), 8.18 (s, 3H), 7.88 (d, J=8.0 Hz, 1H), 7.47 (d, J=7.7 Hz, 1H), 7.36 (d, J=2.6 Hz, 1H), 7.20 (t, J=7.7 Hz, 1H), 4.18 (s, 1H), 3.20-3.32 (2H) partially obscured by water peak. Carboxylic acid OH was not observed. 19F NMR (471 MHz, DMSO-d6) δ −57.79, −74.95. Insufficient quantity for 13C NMR. HRMS (ESI+): for C12H12F3N2O2 [M+H]+ requires 273.0845 found 273.0845.
W-7-CF3 Data
Retention time=16.2 mins. 1H NMR (500 MHz, DMSO-d6) δ 11.73 (d, J=2.6 Hz, 1H), 8.18 (s, 3H), 7.74 (d, J=8.1 Hz, 1H), 7.54 (d, J=2.6 Hz, 1H), 7.46 (d, J=7.4 Hz, 1H), 7.27 (t, J=7.8 Hz, 1H), 4.03 (s, 1H), 3.44-3.38 (m, 1H), 3.13 (dd, J=15.7, 9.6 Hz, 1H). Carboxylic acid OH was not observed 19F NMR (471 MHz, DMSO-d6) δ −61.22, −74.95. Insufficient quantity for 13C NMR. HRMS (ESI+): for C12H12F3N2O2 [M+H]+ requires 273.0845 found 273.0845.
TyrTrp[W-2-CF3]
Synthesised following the general trifluoromethylation procedure 1 on half the scale to obtain a white solid (9.6 mg, 0.0175 mmol, 35%). Reverse phase HPLC details: Phenomenex Synergi™ 4 μm Hydro RP 80 Å, 250×10 mm. Flow rate=4 mL/min. Column temperature=40° C. Eluent=15% MeCN (0.1% TFA) and 85 H2O (0.1% TFA) for 3 mins then increase linearly to 49% MeCN at 25 mins. Retention time=16.3 mins. 1H NMR (500 MHz, DMSO-d6) δ 12.95 (s, 1H), 12.06 (s, 1H), 9.36 (s, 1H), 9.01 (d, J=8.1 Hz, 1H), 8.04 (s, 3H), 7.78 (d, J=8.1 Hz, 1H), 7.44 (d, J=8.2 Hz, 1H), 7.29 (t, J=7.7 Hz, 1H), 7.15 (t, J=7.5 Hz, 1H), 7.06 (d, J=8.4 Hz, 2H), 6.69 (d, J=8.4 Hz, 2H), 4.57 (q, J=7.9 Hz, 1H), 3.89 (d, J=8.9 Hz, 1H), 3.33 (dd, J=14.6, 8.3 Hz, 1H), 3.23 (dd, J=14.6, 6.7 Hz, 1H), 3.02 (dd, J=14.3, 4.9 Hz, 1H), 2.82 (dd, J=14.3, 8.2 Hz, 1H). 13C NMR (126 MHz, DMSO-d6) δ 172.10, 168.11, 156.62, 135.67, 130.54, 126.73, 124.56, 124.36, 123.22 (q, J=269.3 Hz), 121.66 (q, J=36.1 Hz), 120.11, 120.00, 115.36, 112.38, 112.04 (q, J=2.9 Hz), 53.58, 53.37, 36.01, 26.62. 19F NMR (376 MHz, DMSO-d6) δ −57.70, −74.95. HRMS (ESI+): for C21H21O4N3F3 [M+H]+ requires 436.1479 found 436.1480. [α]D25=+29±5° (c 0.071, H2O).
Major Trifluoromethylated Side Products of Tyr-Trp Reaction
Synthesized following the general trifluoromethylation procedure 2: with 0.02 mmol of 70% aqueous TBHP (30 μL of a 2.67M stock solution in 25 mM NH4HCO2), reaction time was 50 mins. The crude reaction mixture was diluted with about 2 mL of 5% MCCN (aq) with 0.1% TFA and each time 0.5 mL was injected into the HPLC loop (1 mL) for purification. The yield is not determined as the amount of product is not enough to be measured accurately on an analytical balance (≤0.1 g). Reverse phase HPLC details: Phenomenex Synergi™ 4 μm Hydro RP 80 Å, 250×10 mm. Flow rate=4 mL/min. Column temperature=40° C. Eluent=15% MCCN (0.1% TFA) and 85 H2O (0.1% TFA) for 3 mins then increase linearly to 49% MeCN at 25 mins. Retention time=19.1 mins (W-4-CF3 and W-7-CF3 are not separated).
W-7-CF3
1H NMR (500 MHz, DMSO-d6) δ 12.91 (s, 1H), 11.32 (d, J=2.6 Hz, 1H), 9.33 (s, 1H), 8.81 (s, 1H), 7.95 (s, 3H), 7.87 (d, J=7.9 Hz, 1H), 7.44 (d, J=7.6 Hz, 1H), 7.31 (d, J=2.5 Hz, 1H), 7.18 (t, J=7.7 Hz, 1H), 7.06 (d, J=11.6 Hz, 2H), 6.69 (d, J=8.6 Hz, 2H), 4.66-4.57 (m, 1H), 3.90 (s, 1H), 3.28-3.14 (m, 2H) partially obscured by HDO peak, 3.12-3.02 (m, 1H), 2.81 (dd, J=14.6, 8.4 Hz, 1H). 19F NMR (471 MHz, DMSO-d6) δ −61.22, −74.95. Insufficient quantity for 13C NMR. HRMS (ESI+): for C21H21O4N3F3[M+H]+ requires 436.1479 found 436.1489.
W-4-CF3
1H NMR (500 MHz, DMSO-d6) δ 12.91 (s, 1H), 11.59 (d, J=2.7 Hz, 1H), 9.34 (s, 1H), 8.93 (d, J=7.9 Hz, 1H), 7.95 (s, 3H), 7.71 (d, J=8.2 Hz, 1H), 7.47-7.40 (m, 2H), 7.24 (t, J=7.8 Hz, 1H), 7.08 (d, J=8.7 Hz, 2H), 6.69 (d, J=8.5 Hz, 2H), 4.66-4.57 (m, 1H), 3.90 (s, 1H), 3.26-3.14 (m, 1H) partially obscured by HDO peak, 3.12-3.02 (m, 2H), 2.81 (dd, J=14.6, 8.4 Hz, 1H). 19F NMR (471 MHz, DMSO-d6) δ −57.63, −74.95. Insufficient quantity for 13C NMR. HRMS (ESI+): for C21H21O4N3F3[M+H]+ requires 436.1479 found 436.1489.
Trp[W-2-CF3]Tyr
Synthesised following the general trifluoromethylation procedure 1 to obtain a white solid (11.0 mg, 0.0200 mmol, 20%). Reverse phase HPLC details: C18(2) Luna, 250×10 mm 5p 100 Å. Flow rate=4 mL/min. Column temperature=25° C. Eluent=19% MeCN (0.1% TFA) in 81% water (0.1% TFA). Retention time=17 mins. 1H NMR (500 MHz, DMSO-d6) δ 12.73 (s, 1H), 12.09 (s, 1H), 9.21 (s, 1H), 8.62 (d, J=7.8 Hz, 1H), 8.27 (s, 3H), 7.79 (d, J=8.1 Hz, 1H), 7.44 (d, J=8.2 Hz, 1H), 7.29 (t, J=7.6 Hz, 1H), 7.15 (t, J=7.5 Hz, 1H), 6.98-6.91 (m, 2H), 6.66-6.59 (m, 2H), 4.37 (dt, J=8.0, 6.3 Hz, 1H), 4.14-4.08 (m, 1H), 3.38-3.29 (m, 1H), 3.17 (dd, J=14.9, 6.8 Hz, 1H), 2.88 (dd, J=13.9, 5.9 Hz, 1H), 2.82 (dd, J=13.9, 6.6 Hz, 1H). 1C NMR (126 MHz, DMSO-d6) δ 171.58, 167.64, 157.68 (q, J=30.6 Hz), 156.06, 135.77, 130.23, 126.78, 126.61, 124.38, 122.63 (q, J=36.0 Hz), 121.89 (q, J=269.2 Hz), 120.12, 120.00, 117.39 (q, J=301.3 Hz), 114.98, 112.38, 109.21 (q, J=2.6 Hz), 54.01, 52.41, 36.31, 26.28. 19F NMR (471 MHz, DMSO-d6) δ −57.59, −74.95. HRMS (ESI+): for C21H21O4N3F3 [M+H]+ requires 436.1479 found 436.1476. [α]D25=+53±1° (c 0.065, H2O).
Major Trifluoromethylated Side Products of Trp-Tyr Reaction
Synthesized following the general trifluoromethylation procedure 2: with 0.02 mmol of 70% aqueous TBHP (30 μL of a 2.67M stock solution in 25 mM NH4HCO2), reaction time was 30 mins. The crude reaction mixture was diluted with about 2 mL of 5% MeCN (aq) with 0.1% TFA and each time 0.5 mL was injected into the HPLC loop (1 mL) for purification. The yield is not determined as the amount of product is not enough to be measured accurately on an analytical balance (≤0.1 g). Reverse phase HPLC details: Phenomenex Synergi™ 4 μm Hydro RP 80 Å, 250×10 mm. Column temperature=25° C. Flow rate=4 mL/min. Column temperature=40° C. Eluent=17% MeCN (0.1% TFA) and 83% H2O (0.1% TFA) for 3 mins then increase linearly to 32% MeCN at 25 mins then another linear increase to 36% MeCN at 27 mins. Retention time=21.6. mins. 1H NMR (500 MHz, DMSO-d6) δ 11.69 (s, 1H), 9.23 (s, 1H), 8.73 (s, 1H), 7.71 (d, J=8.1 Hz, 1H), 7.54 (d, J=2.7 Hz, 1H), 7.43 (d, J=7.5 Hz, 1H), 7.25 (t, J=7.8 Hz, 1H), 7.05-7.00 (m, 2H), 6.69-6.63 (m, 2H), 4.47-4.39 (m, 1H), 4.22 (s, 1H), 3.33-3.07 (m, 2H), 2.96 (dd, J=14.1, 5.3 Hz, 1H), 2.83 (dd, J=14.0, 8.3 Hz, 1H). Protons of NH3 (N-terminal) and the alpha proton adjacent to the NH3 group are not observed probably due to exchange. benzylic protons of phenol are probably obscured by HDO peak. 19F NMR (471 MHz, DMSO-d6) δ −57.30, −74.95. Insufficient quantity for 13C NMR. HRMS (ESI+): for C21H21O4N3F3[M+H]+ requires 436.1479 found 436.1507.
Synthesized following the general trifluoromethylation procedure 2: with 0.02 mmol of 70% aqueous TBHP (30 μL of a 2.67M stock solution in 25 mM NH4HCO2), reaction time was 30 mins. The crude reaction mixture was diluted with about 2 mL of 5% MCCN (aq) with 0.1% TFA and each time 0.5 mL was injected into the HPLC loop (1 mL) for purification. The yield is not determined as the amount of product is not enough to be measured accurately on an analytical balance (≤0.1 g). Reverse phase HPLC details: Phenomenex Synergi™ 4 μm Hydro RP 80 Å, 250×10 mm. Flow rate=4 mL/min. Column temperature=40° C. Eluent=17% MCCN (0.1% TFA) and 83% H2O (0.1% TFA) for 3 mins then increase linearly to 32% MCCN at 25 mins then another linear increase to 36% MeCN at 27 mins. Retention time=24.0. mins. 1H NMR (500 MHz, DMSO-d6) δ 12.98 (s, 1H), 11.41 (s, 1H), 9.25 (s, 1H), 8.92 (s, 1H), 8.03 (d, J=8.1 Hz, 1H), 7.98 (s, 3H), 7.46 (d, J=7.6 Hz, 1H), 7.35 (d, J=2.5 Hz, 1H), 7.19 (t, J=7.7 Hz, 1H), 7.05 (d, J=8.0 Hz, 2H), 6.68 (d, J=8.4 Hz, 2H), 4.47 (q, J=7.2 Hz, 1H), 4.03 (s, 1H), 3.34-3.21 (m, 1H), 3.08 (dd, J=14.9, 9.2 Hz, 1H), 3.05-2.96 (m, 1H) partially obscured by HDO peak, 2.87 (dd, J=14.0, 8.3 Hz, 1H). 19F NMR (471 MHz, DMSO-d6) δ −61.18, −74.95. Insufficient quantity for 13C NMR. HRMS (ESI+): for C21H21O4N3F3[M+H]+ requires 436.1479 found 436.1465.
PheTyr[ortho-CF3]
Synthesised following the general trifluoromethylation procedure 1 to obtain a white solid (34.7 mg, 0.0680 mmol, 68%). Reverse phase HPLC details: C18(2) Luna, 250×10 mm 5μ 100 Å. Flow rate=4 mL/min. Column temperature=25° C. Eluent=28% MeCN (0.1% TFA) and 72% H2O (0.1% TFA). Retention time=10 mins. 1H NMR (500 MHz, DMSO-d6) δ 10.50 (s, 1H), 8.85 (d, J=7.9 Hz, 1H), 8.08 (s, 4H), 7.38 (d, J=2.1 Hz, 1H), 7.35-7.24 (m, 7H), 6.95 (d, J=8.4 Hz, 1H), 4.47 (td, J=7.9, 5.2 Hz, 1H), 4.02 (dd, J=8.5, 4.8 Hz, 1H), 2.91 (ddd, J=14.4, 8.3, 6.2 Hz, 2H). Carboxylic acid-OH is not observed. 13C NMR (126 MHz, DMSO-d6) δ 172.10, 168.20, 157.96, 157.71, 154.58, 134.71, 134.47, 129.56, 128.55, 127.20, 127.16, 127.12, 124.06 (q, J=272.7 Hz), 116.91, 115.09 (q, J=29 Hz), 53.84, 53.16, 36.98, 35.60. 1F NMR (470 MHz, DMSO-d6) δ −62.25, −74.95. HRMS (ESI+): for C19H20O4N2F3 [M+H]+ requires 397.1370 found 397.1371. [α]D25=+28±2° (c 0.044, H2O).
Major Trifluoromethylated Side Products of Phe-Tyr Reaction
Synthesized following the general trifluoromethylation procedure 2: with 0.08 mmol of 70% aqueous TBHP (30 μL of a 2.67M stock solution in 25 mM NH4HCO2), reaction time was 40 mins. The crude reaction mixture was diluted with about 2 mL of 5% MeCN (aq) with 0.1% TFA and each time 0.5 mL was injected into the HPLC loop (1 mL) for purification. The yield is not determined as the amount of product is not enough to be measured accurately on an analytical balance (≤0.1 g). Reverse phase HPLC details: Phenomenex Synergi™ 4 μm Hydro RP 80 Å, 250×10 mm. Column temperature=40° C. Flow rate=4 mL/min. Column temperature=40° C. Eluent=20% MCCN (0.1% TFA) and 80% H2O (0.1% TFA) for 3 mins then increase linearly to 30% MeCN at 25 mins.
Phe[meta-CF3]-Tyr and Phe-Tyr[meta-CF3] (Not separated)
Retention time=18-19.1 mins.
Phe-Tyr[meta-CF3]
1H NMR (500 MHz, DMSO-d6) δ 10.03 (s, 1H), 8.98 (d, J=8.3 Hz, 1H), 8.06 (s, 3H), 7.41-7.21 (m, 6H), 7.05 (d, J=2.7 Hz, 1H), 6.97 (dd, J=8.4, 2.7 Hz, 1H), 4.63-4.38 (m, 1H), 4.00 (dd, J=8.2, 4.9 Hz, 1H), 3.23-3.09 (m, 2H), 3.03-2.85 (m, 2H). Carboxylic acid —OH is not observed. 19F NMR (470 MHz, DMSO-d6) δ −59.96, −74.95. Insufficient quantity for 13C NMR. MS (ESI+): for C19H20O4N2F3 [M+H]+ requires 397.14 found 397.15.
Phe[meta-CF3]-Tyr
1H NMR (500 MHz, DMSO-d6) δ 9.27 (s, 1H), 8.78 (d, J=7.9 Hz, 1H), 8.06 (s, 3H), 7.68 (s, 1H), 7.66-7.62 (m, 1H), 7.61-7.51 (m, 2H), 7.04 (d, J=10.0 Hz, 2H), 6.68 (d, J=8.4 Hz, 2H), 4.56-4.37 (m, 1H), 4.09 (dd, J=8.8, 4.5 Hz, 1H), 3.22-3.11 (m, 2H), 3.05-2.98 (m, 1H), 2.85 (dd, J=14.0, 8.2 Hz, 1H). Carboxylic acid —OH is not observed. 19F NMR (470 MHz, DMSO-d6) δ −62.48, −74.95. Insufficient quantity for 13C NMR. MS (ESI+): for C19H20O4N2F3 [M+H]+ requires 397.14 found 397.15.
NMR Analysis
The chemical shifts of the OH protons of tyrosine provide strong evidence that the phenol group of one of the tyrosine residues is not trifluoromethylated. The main motivation to postulate that this is the Phe-meta-CF3 is that one of the aromatic protons in the 7.5-7.7 ppm region is a broad singlet. This pattern is inconsistent with Phe-ortho-CF3 and Phe-para-CF3. The 13C chemical shifts observed indirectly via HSQC are also consistent with a meta-CF3 on the phenyl group of the Phe residue (refer to the 13C NMR of 3-Trifluoromethyltoluene in Knauber, T. et al., L. J. Chem.—A Eur. J. 2011, 17 (9), 2689-2697).
Phe[para-CF3]-Tyr (Not separated one unidentified impurity with semi-prep column) Retention time=19.4-20.1 mins. 19F NMR (470 MHz, DMSO-d6) δ −62.41, −74.95. HRMS (ESI+): for C19H20O4N2F3 [M+H]+ requires 397.1370 found 397.1388.
NMR Analysis
The chemical shifts of the OH protons of tyrosine residue provide strong evidence that the phenol group of one of the tyrosine residues is not trifluoromethylated. The integration of the amide's NH relative phenol's OH suggested the presence of molecule(s) that does not contain a phenol's OH. Phe[para-CF3]-Tyr is identified by two distinct doublets in the 1H spectrum and their coupling from COSY90. While it is tempting to assign the minor peaks which overlap the two doublets of Phe aromatic protons of Phe[para-CF3]-Tyr to Phe[ortho-CF3]-Tyr, the coupling constant is too small in magnitude to be due to at 3JHH. The 19F chemical shift of Phe[ortho-CF3] is expected to be smaller in magnitude than both meta- and para- as reported in Knauber, T. et al., L. J. Chem.—A Eur. J. 2011, 17 (9), 2689-2697.
PheTrp[W-2-CF3]
Synthesised following the general trifluoromethylation procedure 1 to obtain a white solid (9.5 mg, 0.0178 mmol, 18%). Reverse phase HPLC details: Phenomenex Synergi™ 4 μm Hydro RP 80 Å, 250×10 mm. Flow rate=4 mL/min. Column temperature=40° C. Eluent=15% MCCN (0.1% TFA) and 85% H2O (0.1% TFA) for 3 mins then increase linearly to 49% MCCN at 25 mins. Retention time=18.8 mins. 1H NMR (500 MHz, DMSO-d6) δ 12.92 (s, 1H), 12.07 (s, 1H), 9.04 (d, J=8.2 Hz, 1H), 8.17-8.13 (m, 3H), 7.79 (d, J=8.1 Hz, 1H), 7.45 (d, J=8.3 Hz, 1H), 7.37-7.23 (m, 6H), 7.15 (ddd, J=8.0, 6.8, 1.0 Hz, 1H), 4.57 (td, J=8.2, 6.5 Hz, 1H), 4.00 (d, J=5.6 Hz, 1H), 3.36-3.28 (m, 1H), 3.27-3.19 (m, 1H), 3.13 (dd, J=14.2, 5.2 Hz, 1H), 2.95 (dd, J=14.1, 8.1 Hz, 1H). 13C NMR (126 MHz, DMSO-d6) δ 172.04, 167.98, 135.68, 134.72, 129.54, 128.56, 127.22, 126.73, 124.36, 122.16 (q, J=269.1 Hz), 121.68 (q, J=36.1 Hz), 120.11, 120.00, 112.40, 112.00 (q, J=2.8 Hz), 53.38, 53.32, 36.81, 26.67. 19F NMR (471 MHz, DMSO-d6) δ −57.48, −74.95. HRMS (ESI+): for C21H21F3N3O3 [M+H]+ requires 420.1530 found 420.1527. [α]D25=−71±3° (c 0.051, H2O).
Major Trifluoromethylated Side Products of Phe-Trp Reaction
Synthesized following the general trifluoromethylation procedure 2: with 0.02 mmol of 70% aqueous TBHP (30 μL of a 2.67M stock solution in 25 mM NH4HCO2), reaction time was 50 mins. The crude reaction mixture was diluted with about 2 mL of 5% MCCN (aq) with 0.1% TFA and each time 0.5 mL was injected into the HPLC loop (1 mL) for purification. The collected fractions were lyophilized to give a white solid. The yield is not determined as the amount of product is not enough to be measured accurately on an analytical balance (≤0.1 g). Reverse phase HPLC details: Phenomenex Synergi™ 4 μm Hydro RP 80 Å, 250×10 mm. Flow rate=4 mL/min. Column temperature=40° C. Eluent=15% MeCN (0.1% TFA) and 85% H2O (0.1% TFA) for 3 mins then increase linearly to 49% MCCN at 25 mins. Retention time=21.1 mins. 1H NMR (500 MHz, DMSO-d6) δ 11.31 (s, 1H), 7.85 (d, J=7.9 Hz, 1H), 7.44 (d, J=7.4 Hz, 1H), 7.33-7.22 (m, 6H), 7.17 (t, J=7.6 Hz, 1H), 4.59 (t, J=6.8 Hz, 11H), 3.26-3.10 (m, 2H) Partially obscured by solvent peak. Protons of NH3 (N-terminal) and the alpha proton adjacent to the NH3 group are not observed probably due to exchange. benzylic protons of phenol are probably obscured by HDO peak. 19F NMR (470 MHz, DMSO-d6) δ −61.23, −74.95. Insufficient quantity for 13C NMR. MS (ESI+): for C21H21F3N3O3 [M+H]+ requires 420.15 found 420.15.
Synthesized following the general trifluoromethylation procedure 2: with 0.02 mmol of 70% aqueous TBHP (30 μL of a 2.67M stock solution in 25 mM NH4HCO2), reaction time was 50 mins. The crude reaction mixture was diluted with about 2 mL of 5% MCCN (aq) with 0.1% TFA and each time 0.5 mL was injected into the HPLC loop (1 mL) for purification. The collected fractions were lyophilized to give a white solid. The yield is not determined as the amount of product is not enough to be measured accurately on an analytical balance (≤50.1 g).
Reverse phase HPLC details: Phenomenex Synergi™ 4 μm Hydro RP 80 Å, 250×10 mm. Flow rate=4 mL/min. Column temperature=40° C. Eluent=15% MeCN (0.1% TFA) and 85% H2O (0.1% TFA) for 3 mins then increase linearly to 49% MCCN at 25 mins. Retention time=21.5 mins. 1H NMR (500 MHz, DMSO-d6) δ 11.58 (s, 1H), 7.71 (d, J=8.1 Hz, 1H), 7.43 (d, J=7.5 Hz, 1H), 7.41 (s, 1H), 7.36-7.27 (m, 6H), 7.24 (t, J=7.8 Hz, 1H), 4.63 (q, J=7.5 Hz, 1H), 3.21-2.97 (m, 2H) Partially obscured by solvent peak. Protons of NH3 (N-terminal) and the alpha proton adjacent to the NH3 group are not observed probably due to exchange. benzylic protons of phenol are probably obscured by HDO peak. 19F NMR (470 MHz, DMSO-d6) δ −57.62, −74.95. Insufficient quantity for 13C NMR. HRMS (ESI+): for C21H21F3N3O3 [M+H]+ requires 420.15 found 420.16.
MetTrp[W-2-CF3]
Synthesised following the general trifluoromethylation procedure 1 to obtain a white solid after lyophilization (5.1 mg, 0.00986 mmol, 10%). Reverse phase HPLC details: Phenomenex Synergi™ 4 μm Hydro RP 80 Å, 250×10 mm. Flow rate=4 mL/min. Column temperature=40° C. Eluent=5% MeCN (0.1% TFA) and 95% H2O (0.1% TFA) for 3 mins then increase linearly to 40% MeCN at 25 mins. Retention time=23.0 mins. 1H NMR (500 MHz, DMSO-d6) δ 12.72 (s, 1H), 12.07 (s, 1H), 8.96 (d, J=8.0 Hz, 1H), 8.20 (s, 3H), 7.76 (d, J=8.1 Hz, 1H), 7.44 (dd, J=8.3, 1.0 Hz, 1H), 7.29 (ddd, J=8.2, 6.9, 1.1 Hz, 1H), 7.14 (ddd, J=8.1, 6.9, 1.0 Hz, 1H), 4.54 (q, J=7.6 Hz, 1H), 3.80 (d, J=6.3 Hz, 1H), 3.36-3.30 (m, 1H), 3.26-3.18 (m, 1H), 2.53-2.42 (m, 2H), 2.05 (s, 3H), 2.04-1.96 (m, 1H). 13C NMR (126 MHz, DMSO-d6) δ 172.19, 168.15, 135.65, 126.75, 124.34, 122.17 (q, J=269.1 Hz), 121.63 (q, J=36.2 Hz), 120.11, 119.98, 112.38, 112.20 (q, J=2.9 Hz), 53.51, 51.51, 30.89, 27.88, 26.07, 14.43. 19F NMR (470 MHz, DMSO-d6) δ −57.71, −74.95. HRMS (ESI+): for C17H21F3N3O332S [M+H]+ requires 404.1250 found 404.1253. [α]D25=+18±1° (c 0.076, H2O).
Major Trifluoromethylated Side Products of Met-Trp Reaction
Synthesized following the general trifluoromethylation procedure 2 but with 0.01 mmol of substrate (all reagents were halved): with 0.02 mmol of 70% aqueous TBHP (10.7 μL of a 2.67M stock solution in 25 mM NH4HCO2), reaction time was 25 mins. The crude reaction mixture was diluted with about 0.9 mL of 5% MeCN (aq) with 0.1% TFA and injected into the HPLC loop (1 mL) for purification. Reverse phase HPLC details: Phenomenex Synergi™ 4 μm Hydro RP 80 Å, 250×10 mm. Flow rate=4 mL/min. Column temperature=40° C. Eluent=5% MeCN (0.1% TFA) and 95% H2O (0.1% TFA) for 3 mins then increase linearly to 40% MeCN at 25 mins. Retention time=25.6 mins. (W-4-CF3 and W-7-CF3 are not separated)
W-7-CF3
1H NMR (500 MHz, DMSO-d6) δ 12.97 (s, 1H), 11.32 (d, J=2.6 Hz, 1H), 8.75 (d, J=7.5 Hz, 1H), 8.10 (s, 3H), 7.87 (d, J=8.0 Hz, 1H), 7.45 (d, J=7.0 Hz, 1H), 7.32 (d, J=2.5 Hz, 1H), 7.18 (t, J=7.7 Hz, 1H), 4.59 (dtd, J=13.0, 8.0, 4.8 Hz, 1H), 4.03-3.69 (m, 1H), 3.36-2.92 (m, 2H), 2.52 (dd, J=4.8, 2.9 Hz, 1H), 2.13-1.92 (m, 6H). 19F NMR (376 MHz, DMSO-d6) δ −61.20, −74.95. Insufficient quantity for 13C NMR. MS (ESI+): for C17H21F3N3O332S [M+H]+ requires 404.13 found 404.13.
W-4-CF3
1H NMR (500 MHz, DMSO-d6) δ 12.89 (s, 1H), 11.59 (d, J=2.6 Hz, 1H), 8.87 (d, J=7.8 Hz, 1H), 8.10 (s, 3H), 7.71 (d, J=8.1 Hz, 1H), 7.43 (d, J=7.2 Hz, 1H), 7.40 (d, J=2.6 Hz, 1H), 7.24 (t, J=7.8 Hz, 1H), 4.59 (dtd, J=13.0, 8.0, 4.8 Hz, 1H), 4.03-3.69 (m, 1H), 3.12 (ddd, J=40.7, 15.3, 9.3 Hz, 2H), 2.52 (dd, J=4.8, 2.9 Hz, 1H), 2.13-1.92 (m, 6H). 19F NMR (376 MHz, DMSO-d6) δ −57.61, −74.95. Insufficient quantity for 13C NMR. MS (ESI+): for C17H21F3N3O332S [M+H]+ requires 404.13 found 404.13.
MetTyr[ortho-CF3]
Synthesised following the general trifluoromethylation procedure to obtain a white solid after lyophilization (4.9 mg, 0.00981 mmol, 10%). Reverse phase HPLC details: Phenomenex Synergi™ 4 μm Hydro RP 80 Å, 250×10 mm. Flow rate=4 mL/min. Column temperature=40° C. Eluent=5% MeCN (0.1% TFA) and 85% H2O (0.1% TFA) for 3 mins then increase linearly to 30% MeCN at 30 mins. Retention time=24.9 mins. 1H NMR (500 MHz, DMSO-d6) δ 12.99 (s, 1H), 10.51 (s, 1H), 8.76 (d, J=7.6 Hz, 1H), 8.16 (s, 3H), 7.39 (d, J=2.2 Hz, 1H), 7.33 (dd, J=8.5, 2.2 Hz, 1H), 6.95 (d, J=8.3 Hz, 1H), 4.53-4.33 (m, 1H), 3.83 (d, J=7.3 Hz, 1H), 3.05 (dd, J=14.2, 4.9 Hz, 1H), 2.89 (dd, J=14.2, 9.1 Hz, 1H), 2.49 (s, 2H), 2.04 (s, 5H). 13C NMR (126 MHz, DMSO-d6) δ 172.27, 168.32, 154.56, 134.35, 127.33, 127.09 (q, J=4.7 Hz), 124.09 (q, J=272.0 Hz), 116.92, 115.10 (q, J=29.7 Hz), 53.98, 51.45, 35.18, 31.10, 27.81, 14.42. 19F NMR (470 MHz, DMSO-d6) δ−62.34, −74.95. HRMS (ESI+): for C15H20F3N2O431S [M+H]+ requires 381.1090 found 381.1088. [α]D25=483° (c 0.073, H2O).
Major Trifluoromethylated Side Products of Met-Tyr Reaction
Synthesized following the general trifluoromethylation procedure 2: with 0.04 mmol of 70% aqueous TBHP (15 μL of a 2.67M stock solution in 25 mM aq. NH4HCO2), reaction time was 50 mins. The crude reaction mixture was diluted with about 2 mL of 10% MeCN (aq) with 0.1% TFA and each time 0.5 mL was injected into the HPLC loop (1 mL) for purification. The yield is not determined as the amount of product is not enough to be measured accurately on an analytical balance (≤0.1 g). Reverse phase HPLC details: Phenomenex Synergi™ 4 μm Hydro RP 80 Å, 250×10 mm. Flow rate=4 mL/min. Column temperature=40° C.
Eluent=5% MeCN (0.1% TFA) and 95% H2O (0.1% TFA) for 3 mins then increase linearly to 30% MeCN at 30 mins. Retention time=24.3 mins. 1H NMR (500 MHz, DMSO-d6) δ 8.86 (d, J=8.2 Hz, 1H), 8.22-7.95 (m, 3H), 10.00 (s, 1H), 7.30 (d, J=8.4 Hz, 1H), 7.05 (d, J=2.6 Hz, 1H), 6.96 (dd, J=8.5, 2.7 Hz, 1H), 4.45 (ddd, J=9.6, 8.1, 5.4 Hz, 1H), 3.88-3.77 (m, 1H), 3.22 (dd, J=15.0, 5.5 Hz, 1H)partially obscured by HDO peak, 2.94 (dd, J=14.7, 9.8 Hz, 1H), 2.52-2.51 (m, 2H) overlap with solvent peak, 2.06 (s, 3H), 2.05-1.94 (m, 2H). 19F NMR (470 MHz, DMSO-d6) δ −59.95, −74.95. Insufficient quantity for 13C NMR. MS (ESI+): for C15H20F3N2O432S [M+H]+ requires 381.11 found 381.11.
Synthesized following the general trifluoromethylation procedure 2: with 0.04 mmol of 70% aqueous TBHP (15 μL of a 2.67M stock solution in 25 mM NH4HCO2), reaction time was 50 mins. The crude reaction mixture was diluted with about 2 mL of 10% MeCN (aq) with 0.1% TFA and each time 0.5 mL was injected into the HPLC loop (1 mL) for purification. The yield is not determined as the amount of product is not enough to be measured accurately on an analytical balance (≤0.1 g). Reverse phase HPLC details: Phenomenex Synergi™ 4 μm Hydro RP 80 Å, 250×10 mm. Flow rate=4 mL/min. Column temperature=40° C. Eluent=5% MCCN (0.1% TFA) and 95% H2O (0.1% TFA) for 3 mins then increase linearly to 30% MCCN at 30 mins. Retention time=21.3 mins. 1H NMR (500 MHz, DMSO-d6) δ 10.41 (s, 1H), 8.76 (s, 1H), 7.39 (s, 1H), 7.32 (d, J=8.3 Hz, 1H), 6.94 (d, J=8.4 Hz, 1H), 4.46 (q, J-8.8, 7.4 Hz, 1H), 3.89 (s, 1H), 3.05 (dd, J=14.2, 4.9 Hz, 1H), 2.97-2.85 (m, 2H), 2.86-2.72 (m, 1H), 2.54 (s, 3H)partially obscured by solvent peak, 2.15-1.93 (m, 2H). Cosy does not provide correlation, probably due to exchange at the NHs group. Carboxylic acid proton and NH3 protons were not observed. 19F NMR (471 MHz, DMSO-d6) δ −62.32, −62.33, −74.95. CFs is observed as two singlets of similar chemical shifts probably due to the new chiral centre at the sulfoxide. MS (ESI+): for C15H20F3N2O532S [M+H]+ requires 397.10 found 397.10.
Synthesised following the general trifluoromethylation procedure to obtain a white solid after lyophilization (7.0 mg, 0.0114 mmol, 11%). Reverse phase HPLC details: Phenomenex Synergi™ 4 μm Hydro RP 80 Å, 250×10 mm. Flow rate=4 mL/min. Column temperature=40° C. Eluent=5% MeCN (0.1% TFA) and 95% H2O (0.1% TFA) for 3 mins then increase linearly to 25% MeCN at 25 mins. Retention time=17.3 mins. 1H NMR (500 MHz, DMSO-d6) δ 14.41 (s, 2H), 10.58 (s, 1H), 9.01 (d, J=8.1 Hz, 2H), 8.10 (s, 3H), 7.43 (t, J=2.8 Hz, 2H), 7.32 (dd, J=8.5, 2.2 Hz, 1H), 6.95 (d, J=8.4 Hz, 1H), 4.65 (td, J=8.1, 5.2 Hz, 1H), 4.02 (t, J=6.4 Hz, 1H), 3.20 (dd, J=15.2, 5.3 Hz, 1H), 3.07 (td, J=12.7, 10.8, 6.7 Hz, 2H), 2.88 (dd, J=14.4, 8.3 Hz, 1H). Carboxylic acid OH is not observed. 13C NMR (126 MHz, DMSO-d6) δ 171.38, 168.22, 155.08, 134.89, 134.04, 128.94, 127.65 (q, J=4.3, 3.8 Hz), 124.62, 124.05 (q, J=272.5 Hz), 117.12, 117.03, 115.42 (q, J=30.0 Hz), 53.33, 51.36, 35.73, 26.32. 19F NMR (470 MHz, DMSO-d6) δ −62.22, −74.95. HRMS (ESI+): for C16H18O4N4F3[M+H]+ requires 387.1275 found 387.1278. [α]D25=+28±1° (c 0.073, H2O).
Major Trifluoromethylated Side Products of Tyr-His Reactions
Synthesized following the general trifluoromethylation procedure 2: with 0.08 mmol of 70% aqueous TBHP (30 μL of a 2.67M stock solution in 25 mM NH4HCO2), reaction time was 50 mins. The crude reaction mixture was diluted with about 2 mL of 5% MeCN (aq) with 0.1% TFA and each time 0.5 mL was injected into the HPLC loop (1 mL) for purification. The yield is not determined as the amount of product is not enough to be measured accurately on an analytical balance (≤0.1 g). Reverse phase HPLC details: Phenomenex Synergi™ 4 μm Hydro RP 80 Å, 250×10 mm. Flow rate=4 mL/min. Column temperature=40° C. Eluent=5% MCCN (0.1% TFA) and 95% H2O (0.1% TFA) for 3 mins then increase linearly to 25% MCCN at 25 mins. Retention time=16.1 mins. 1H NMR (500 MHz, DMSO-d6) δ 10.07 (s, 1H), 8.75 (d, J=7.9 Hz, 1H), 8.28 (s, 3H), 7.25 (d, J=8.5 Hz, 1H), 7.05 (d, J=2.6 Hz, 1H), 6.96 (dd, J=8.4, 2.6 Hz, 1H), 5.32 (t, J=5.0 Hz, 1H), 4.60 (q, J=7.3 Hz, 1H), 3.17-2.92 (m, 41H). Imidazole's proton (3H) and CO2H (1H) were not observed. 19F NMR (471 MHz, DMSO-d6) δ −59.68, −74.95. Insufficient quantity for 13C NMR. HRMS (ESI+): for C16H18O4N4F3 [M+H]+ requires 387.1275 found 387.1270.
HisTrp[W-2-CF3]
Synthesised following the general trifluoromethylation procedure to obtain a white solid (7.4 mg, 0.0116 mmol, 12%). Reverse phase HPLC details: Phenomenex Synergi™ 4 μm Hydro RP 80 Å, 250×10 mm. Flow rate=4 mL/min. Column temperature=40° C. Eluent=11% MCCN (0.1% TFA) and 89% H2O (0.1% TFA) for 3 mins then increase linearly to 25% MeCN at 25 mins, then another linear increase to 40% at 30 mins. Retention time=20.6 mins. 1H NMR (500 MHz, DMSO-d6) δ 14.39 (s, 2H), 12.07 (s, 1H), 9.02-8.97 (m, 2H), 8.38-8.33 (m, 3H), 7.77 (d, J=8.1 Hz, 1H), 7.45 (d, J=8.3 Hz, 1H), 7.42 (s, 1H), 7.32-7.24 (m, 1H), 7.14 (t, J=7.5 Hz, 1H), 4.56 (td, J=8.1, 6.7 Hz, 1H), 4.14 (t, J=6.7 Hz, 1H), 3.31 (dd, J=14.6, 8.3 Hz, 1H), 3.27-3.20 (m, 2H), 3.16 (dd, J=15.6, 7.3 Hz, 1H).
Carboxylic acid OH is not observed. 13C NMR (126 MHz, DMSO-d6) δ 172.10, 167.29, 135.69, 134.45, 126.71, 124.39, 122.17 (q, J=268.9 Hz), 121.70 (q, J=36.1 Hz), 120.14, 119.94, 118.00, 112.43, 112.01 (q, J=2.9 Hz), 53.59, 51.22, 26.45, 26.37. 19F NMR (470 MHz, DMSO-d6) δ −57.37, −74.95. HRMS (ESI+): for C18H19F3N5O3[M+H]˜ requires 410.1434 m/z found 410.1429 m/z. [α]D25=+10±2° (c 0.119, H2O).
Major Trifluoromethylated Side Products of his-Trp Reactions
Synthesized following the general trifluoromethylation procedure 2: with 0.08 mmol of 70% aqueous TBHP (30 μL of a 2.67M stock solution in 25 mM NH4HCO2), reaction time was 50 mins. The crude reaction mixture was diluted with about 2 mL of 5% MCCN (aq) with 0.1% TFA and each time 0.5 mL was injected into the HPLC loop (1 mL) for purification. The yield is not determined as the amount of product is not enough to be measured accurately on an analytical balance (≤0.1 g). Reverse phase HPLC details: Phenomenex Synergi™ 4 μm Hydro RP 80 Å, 250×10 mm. Flow rate=4 mL/min. Column temperature=40° C. Eluent=11% MCCN (0.1% TFA) and 89% H2O (0.1% TFA) for 3 mins then increase linearly to 25% MeCN at 25 mins, then another linear increase to 40% at 30 mins. Retention time=28.1 mins.
W-4-CF3
1H NMR (500 MHz, DMSO-d6) δ 11.58 (d, J=2.7 Hz, 1H), 9.01 (s, 1H), 8.18 (s, 3H), 7.71 (d, J=8.2 Hz, 1H), 7.43 (d, J=7.3 Hz, 1H), 7.40 (d, J=2.6 Hz, 1H), 7.24 (t, J=7.8 Hz, 1H), 4.60 (d, J=8.4 Hz, 1H), 4.07 (s, 1H), 3.28-2.94 (m, 2H). Imidazole's protons are not observed. 8 protons of Histidine are not observed. Carboxylic acid proton is not observed. Insufficient quantity for 13C NMR. 19F NMR (470 MHz, DMSO-d6) δ −57.59, −74.95. HRMS (ESI+): for C15H19F3N5O3[M+H]+ requires 410.1434 m/z found 410.1452 m/z.
W-7-CF3
1H NMR (500 MHz, DMSO-d6) δ 11.37-11.26 (m, 1H), 9.01 (s, 2H), 8.18 (s, 3H), 7.87 (d, J=8.0 Hz, 1H), 7.44 (d, J=6.9 Hz, 1H), 7.31 (d, J=2.5 Hz, 1H), 7.18 (t, J=7.7 Hz, 1H), 4.65-4.50 (m, 1H), 4.07 (s, 1H), 3.27-2.96 (m, 2H). Imidazole's protons are not observed. β protons of Histidine are not observed. Carboxylic acid proton is not observed. Insufficient quantity for 13C NMR. 19F NMR (470 MHz, DMSO-d6) δ −61.18, −74.95. HRMS (ESI+): for C18H19F3N5O3 [M+H]+ requires 410.1434 m/z found 410.1452 m/z.
GluTrp[W-2-CF3]
To GluTrp (10.4 mg, 31 μmol) in a 3 mL screw top V-Vials® with open-top cap (Sigma-Aldrich Z115142) was added 90 μL of DMSO, Iron (III) Chloride (5.5 mg, 34 μmol), NaSO2CF3 (45 μmol, 45 μL of a 1M stock solution in 25 mM aqueous NH4HCO2), and 70% TBHP in water (84 μmol, 45 μL of a 1.87M stock solution in 25 mM aqueous NH4HCO2). The vial was placed in a water bath at 40° C. and stirred at 760 rpm for 30 mins. The crude reaction mixture was diluted with about 2 mL of 5% MeCN (aq) with 0.1% TFA and each time 0.5 mL was injected into the HPLC loop (1 mL) for purification. A white solid which slowly turns brown at −20° C. was obtained after lyophilization (4.6 mg, 3.4 μmol, 11%). Reverse phase HPLC details: Phenomenex Synergi™ 4 μm Hydro RP 80 Å, 250×10 mm. Flow rate=4 mL/min. Column temperature=40° C. Eluent=12% MeCN (0.1% TFA) and 88% H2O (0.1% TFA) for 3 mins then increase linearly to 40% MeCN at 25 mins. Retention time=17.7 mins. 1H NMR (500 MHz, DMSO-d6) δ 12.90 (s, 1H), 12.05 (s, 1H), 8.94 (d, J=8.0 Hz, 1H), 8.12 (d, J=5.3 Hz, 3H), 7.76 (d, J=8.1 Hz, 1H), 7.44 (d, J=8.3 Hz, 1H), 7.29 (t, J=7.6 Hz, 1H), 7.14 (t, J=7.5 Hz, 1H), 4.54 (q, J=7.6 Hz, 1H), 3.78 (q, J=5.7 Hz, 1H), 3.34 (dd, J=14.5, 7.9 Hz, 1H), 3.22 (dd, J=14.8, 7.1 Hz, 1H), 2.38-2.31 (m, 2H), 1.98 (dt, J=9.4, 6.7 Hz, 2H). 13C NMR (126 MHz, DMSO-d6) δ 173.38, 172.18, 168.29, 158.85-156.88 (m), 135.63, 126.73, 124.35, 122.15 (q, J=268.7 Hz), 121.62 (q, J=36.1 Hz), 120.12, 119.97, 116.73 (q, J=297.1 Hz), 112.36, 112.15 (q, J=2.7 Hz), 53.50, 51.45, 28.84, 26.33, 26.15. 19F NMR (471 MHz, DMSO-d6) δ −57.32, −74.95. HRMS (ESI+): for C17H19O5N3F3[M+H]+ requires 402.1271 m/z found 402.1268 m/z. [α]D25=+14±2° (c 0.040, H2O).
Major Trifluoromethylated Side Products of Glu-Trp Reactions
Reverse phase HPLC details: Phenomenex Synergi™ 4 μm Hydro RP 80 Å, 250×10 mm. Flow rate=4 mL/min. Column temperature=40° C. Eluent=12% MeCN (0.1% TFA) and 88% H2O (0.1% TFA) for 3 mins then increase linearly to 40% MCCN at 25 mins. Retention time 21.4 mins. (4-CF3 and 7-CF3 are not separated).
4-CF3
1H NMR (500 MHz, DMSO-d6) δ 12.33 (s, 1H), 11.64-11.51 (m, 1H), 8.87 (d, J=7.7 Hz, 1H), 8.09 (s, 3H), 7.71 (d, J=8.1 Hz, 1H), 7.43 (d, J=7.3 Hz, 1H), 7.41 (d, J=2.6 Hz, 2H), 7.24 (t, J=7.8 Hz, 1H), 4.69-4.46 (m, 1H), 3.82 (s, 1H), 3.48-2.99 (m, 2H), 2.07-1.91 (m, 4H). 19F NMR (471 MHz, DMSO-d6) δ −57.66, −61.23, −74.95. Insufficient quantity for 13C NMR. HRMS (ESI+): for C17H19O5N3F3[M+H]+ requires 402.1271 m/z found 402.1279 m/z.
7-CF3
1H NMR (500 MHz, DMSO-d6) δ 12.88 (s, 2H), 11.32 (d, J=2.1 Hz, 1H), 8.76 (d, J=7.7 Hz, 1H), 8.09 (s, 3H), 7.87 (d, J=7.9 Hz, 1H), 7.45 (d, J=6.8 Hz, 1H), 7.32 (d, J=2.1 Hz, 1H), 7.18 (t, J=7.7 Hz, 1H), 4.69-4.46 (m, 1H), 3.82 (s, 1H), 3.36-2.99 (m, 2H), 2.07-1.91 (m, 4H). 19F NMR (471 MHz, DMSO−d6) δ −57.66, −61.23, −74.95. Insufficient quantity for 13C NMR. HRMS (ESI+): for C17H19O5N3F3[M+H]+ requires 402.1271 m/z found 402.1279 m/z.
Angiotensin(1-7)[Y-ortho-CF3]
To Angiotensin (1-7) (6.3 mg, 5.1 μmol) in a 3 mL screw top V-Vials® with open-top cap (Sigma-Aldrich Z115142) was added 20 μL of DMSO, Iron (III) Chloride (4.4 mg, 27 μmol), NaSO2CF3 (10 μmol, 10 μL of a 1M stock solution in 25 mM aqueous NH4HCO2), and 70% TBHP in water (19 μmol, 10 μL of a 1.87M stock solution in 25 mM aqueous NH4HCO2). The vial was placed in a water bath at 40° C. and stirred at 760 rpm for 50 mins. The crude reaction mixture was diluted with about 2 mL of 10% MeCN (aq) with 0.1% TFA and each time 0.5 mL was injected into the HPLC loop (1 mL) for purification. A white solid was obtained after lyophilization (2.4 mg, 1.8 μmol, 36%). Reverse phase HPLC details: Phenomenex Synergi™ 4 μm Hydro RP 80 Å, 250×10 mm. Flow rate=4 mL/min. Column temperature=40° C. Eluent=8% MeCN (0.1% TFA) and 92% H2O (0.1% TFA) for 3 mins then increase linearly to 30% MeCN at 30 mins. Retention time 25.9 mins. 1H NMR (500 MHz, DMSO-d6) Only Tyrosine residue protons are listed. 7.41 (d, J=2.1 Hz, 1H), 7.31 (dd, J=8.6, 2.1 Hz, 1H), 7.23 (s, 2H), 19F NMR (471 MHz, DMSO-d6) δ −62.17, −74.95. HRMS (ESI+): for C42H62O11N12F3[M+H]+ requires 967.4608 found 967.4606.
Major Trifluoromethylated Side Product of Angiotensin(1-7) Reaction
Reverse phase HPLC details: Phenomenex Synergi™ 4 μm Hydro RP 80 Å, 250×10 mm. Flow rate=4 mL/min. Column temperature=40° C. Eluent=8% MeCN (0.1% TFA) and 92% H2O (0.1% TFA) for 3 mins then increase linearly to 30% MeCN at 30 mins. Retention time 25.9 mins. 19F NMR (471 MHz, DMSO-d6) δ −60.03, −74.95. HRMS (ESI+): for C42H62O11N12F3[M+H]+ requires 967.4608 found 967.4641.
Melittin[W-2-CF3]
To Melittin (0.7 mg, 0.25 μmol) in a 3 mL screw top V-Vials® with open-top cap (Sigma-Aldrich Z115142) was added 25 μL of DMSO, Iron (III) Chloride (3.8 μmol, 5 μL of a 0.76M stock solution in 25 mM aqueous NH4HCO2), NaSO2CF3 (1.9 μmol, 5 μL of a 0.38M stock solution in 25 mM aqueous NH4HCO2), and 70% TBHP in water (3.5 μmol, 5 μL of a 0.70M stock solution in 25 mM aqueous NH4HCO2). Additional 10 μL of 25 mM NH4HCO2 (aq) was added. The vial was placed in a water bath at 40° C. and stirred at 760 rpm for 20 mins. About 1 mL of 15% MeCN (aq) with 0.1% TFA was added to quench the reaction for semi-preparative HPLC. The yield is not determined as the amount of product is not enough to be measured accurately on an analytical balance (≤0.1 g). Reverse phase HPLC details: Zorbax 300SB-C18, 9.4×250 mm 5p 300 Å. Flow rate=4 mL/min. Column temperature=40° C. Eluent=17.5% MeCN (0.1% TFA) and 82.5 water (0.1% TFA) for 6 mins and increase linearly to 55% at 25 mins. Retention time=23.3 mins. 1H NMR (500 MHz, D2O with 0.1% CF3CO2D) Only Trpytophan residue 1H is listed, absence of singlet supported the position of CF3. δ7.74 (d, J=8.0 Hz, 1H), 7.53 (d, J=8.6 Hz, 1H), 7.38 (t, J=8.1 Hz, 1H), 7.22 (t, J=7.6 Hz, 1H). 19F NMR (470 MHz, D2O with 0.1% CF3CO2D) δ −57.87, −75.50. HRMS (ESI+): for C132H232O31N39F3 [M+4H]4+ requires 729.1927 found 729.1926.
Somatostatin-14[W-2-CF3]
To Somatostatin-14 (2.4 mg, 1.5 μmol) in a 3 mL screw top V-Vials® with open-top cap (Sigma-Aldrich Z115142) was added 40 μL of DMSO, Iron (III) Chloride (7.6 μmol, 10 μL of a 0.76M stock solution in 25 mM aqueous NH4HCO2), NaSO2CF3 (5.7 μmol, 15 μL of a 0.38M stock solution in 25 mM aqueous NH4HCO2), and 70% TBHP in water (7.0 μmol, 10 μL of a 0.70M stock solution in 25 mM aqueous NH4HCO2). The vial was placed in a water bath at 40° C. and stirred at 760 rpm for 25 mins. About 1 mL of 15% MeCN (aq) with 0.1% TFA was added to quench the reaction for semi-preparative HPLC. The yield is not measured as the amount of product is not enough to be measured accurately on an analytical balance (≤0.1 g). Reverse phase HPLC details: Zorbax 300SB-C18, 9.4×250 mm 5p 300 Å. Flow rate=4 mL/min. Column temperature=40° C. Eluent=17.5% MeCN (0.1% TFA) in water (0.1% TFA) for 6 mins and increase linearly to 47% at 25 mins. Retention time=19.4 mins. 19F NMR (470 MHz, DMSO-d6) δ −57.58, −74.95. HRMS (ESI+): for C77H10O19N19F332S2[M+H]+ requires 1705.7113 found 1705.7086.
Endomorphin 1[W-2-CF3]
To Endomorphin 1 (5.9 mg, 9.7 μmol) in a 3 mL screw top V-Vials® with open-top cap (Sigma-Aldrich Z115142) was added 40 μL of DMSO, Iron (III) Chloride (20 μmol, 20 μL of a 1M stock solution in 25 mM aqueous NH4HCO2), NaSO2CF3 (15 μmol, 15 μL of a 1M stock solution in 25 mM aqueous NH4HCO2), and 70% TBHP in water (15 μmol, 15 μL of a 1M stock solution in 25 mM aqueous NH4HCO2). The vial was placed in a water bath at 40° C. and stirred at 760 rpm for 40 mins. About 1 mL of 15% MCCN (aq) with 0.1% TFA was added to quench the reaction for semi-preparative HPLC. 0.59 mg (7% yield) of white solid was obtained after lyophilization. Reverse phase HPLC details: Synergi RP 4, 10.0×250 mm 5p 80 Å. Column temperature=40° C. Flow rate=4 mL/min. Eluent=17% MeCN (0.1% TFA) in water (0.1% TFA) for 3 mins and increase linearly to 48% at 25 mins. Retention time=19.8 mins. 1H NMR (500 MHz, DMSO-d6) δ 11.91 (s, 1H), 9.33 (s, 1H), 8.08 (d, J=8.1 Hz, 1H), 7.99 (s, 3H), 7.83 (d, J=8.1 Hz, 1H), 7.74 (d, J=8.1 Hz, 1H), 7.40 (d, J=8.2 Hz, 1H), 7.33-7.18 (m, 3H), 7.18-7.07 (m, 7H), 6.99 (s, 1H), 6.68 (d, J=8.5 Hz, 2H), 4.61-4.46 (m, 1H), 4.45-4.32 (m, 1H), 4.22 (s, 1H), 3.70-3.50 (m, 2H), 3.29-3.19 (m, 1H), 3.17-3.03 (m, 2H), 2.95 (td, J=14.7, 14.2, 5.8 Hz, 2H), 2.89-2.73 (m, 2H), 2.04-1.91 (m, 1H), 1.84-1.58 (m, 3H). Minor conformers peaks are not included. 19F NMR (470 MHz, DMSO-d6) δ −57.51, −74.95. HRMS (ESI+): for C35H38O5N6F3[M+H]+ requires 679.2850 found 679.2847.
NMR Assignment
Assignment of 1H NMR is complicated by the presence of cis- and trans-conformer (28%/72%, determined from 1H NMR by the integration of phenol OH's protons). This is also observed for Endomorphin 1 (cis- and trans-ratio of 25%/75%) as reported by Podlogar et al. (Podlogar, B. L.; Paterlini, M. G.; Ferguson, D. M.; Leo, G. C.; Demeter, D. A.; Brown, F. K.; Reitz, A. B. FEBS Lett. 1998, 439 (1-2), 13-20).
Major Trifluoromethylated Side Products of Endomorphin 1 Reaction
Assignment of structure is based on 19F NMR chemical shift comparison with previous Trp-containing dipeptides and L-Tryptophan. TOCSY revealed coupling between indole NH and C2-H thus supporting that CF3 is not on C2. MS/MS analysis. 4 indole's NHs are due to cis- and trans-isomer. 19F NMR (470 MHz, DMSO-d6) δ −57.22, −57.24, −61.08, −61.10, −74.95. MS (ESI+): for C35H38O5N6F3[M+H]+ requires 679.29 found 679.31.
Insulin
Synthesized according to the procedure of Krska et al(Ichiishi, N.; Caldwell, J. P.; Lin, M.; Zhong, W.; Zhu, X.; Streckfuss, E. C.; Kim, H.-Y. Y.; Parish, C. A.; Krska, S. W. Chem. Sci. 2018, 9 (17), 4168-4175) except that no precaution was taken to exclude oxygen and water. The reaction was done on a smaller scale of 20 mg of recombinant insulin. Purification was performed by reversed phase HPLC. Reverse phase HPLC details: Zorbax 300SB-C18, 9.4×250 mm 5μ 300 Å. Flow rate=4 mL/min. Column temperature=40° C. Eluent=24% MeCN (0.1% TFA) and 76% H2O (0.1% TFA) for 3 mins and increase linearly to 42% MeCN at 25 mins.
Insulin Chain A Y14-CF3
2.9 mg of white solid was obtained after lyophilization. Retention time=19.1 mins. 19F NMR (471 MHz, Deuterium Oxide+0.1% (v/v) Trifluoroacetic Acid-d1) δ −62.06, −75.51. MS data is shown in
Insulin Chain A Y19-CF3
2.9 mg of white solid was obtained after lyophilization. Retention time=18.0 mins. 19F NMR (471 MHz, Deuterium Oxide+0.1% (v/v) Trifluoroacetic Acid-d1) δ −60.91, −75.51. MS data is shown in
Insulin Chain B Y16-CF3 and Insulin Chain B Y26-CF3
1.7 mg of white solid was obtained after lyophilization. Y16-CF3 and Y26-CF3 are separable to a certain extent, however, for convenience, they were collected together for NMR measurement. As LC-MS/MS requires a lower amount of analyte, a small amount of these were separately purified for LC-MS/MS. 19F NMR (471 MHz, Deuterium Oxide+0.1%(v/v) Trifluoroacetic Acid-d1) δ −61.92, −61.95, −75.51. MS data is shown in
Radiochemistry
General Experimental Details
18F-Fluoride was produced by Alliance Medical (UK) via the 18O(p,n) 18F reaction and delivered as 18F-fluoride in 18O-water. Radiosynthesis and azeotropic drying was performed on a NanoTek® automated microfluidic device (Advion). HPLC analysis was performed with a Dionex Ultimate 3000 dual channel HPLC system equipped with shared autosampler, parallel UV-detectors and LabLogic NaI/PMT-radiodetectors with Flowram analog output. Radio-TLC was performed on Merck Kiesegel 60 F254 plates. Analysis was performed using a plastic scintillator/PMT detector. Mass-spec analysis (ESI) of crude reaction mixtures was performed by diverting part of the HPLC flow after passing through the radiodetector to the inlet of an Advion CMS. This enabled the detection of non-labelled 19F-compounds formed during the reaction (the amount of 18F-labelled compounds formed is below the limit of detection). Due to the separation of the modules the radio-signal is offset by 0.1-0.3 min from the UV signal.
HPLC Eluents—Eluent A
HPLC gradient: MeCN/H2O 25 mM NH4HCO2, 1 mL/min, Synergi 4 μm Hydro-RP 80 Å column, 150×4.6 mm
0-1 min (1% MeCN) isocratic
1-10 min (1% MeCN to 95% MeCN) linear increase
10-14 min (95% MeCN) isocratic
14-16 min (95% MeCN to 1% MeCN) linear decrease
16-18 min (5% MeCN) isocratic
Sep-Pak Cartridges Used for Purification
Waters Sep-Pak Al2O3 N light cartridge (part #WAT023561), Waters Sep-Pak SiO2 light cartridge (part #WAT023537), Waters Sep-Pak SiO2 plus cartridge (part #WAT020520), Waters Sep-Pak Dry Sodium Sulfate cartridge (part #WAT054265), Oasis MCX Plus cartridge (Waters, part #186003516), Oasis HLB Plus cartridge (Waters, part #186000132), Oasis HLB Light cartridge (Waters, part #186005125), Oasis MAX Plus cartridge (Waters, part #186003517). All cartridges were preconditioned with MeOH (2 mL) followed by H2O (10 mL) unless otherwise indicated.
General Procedure for the Small Scale 18F-Labelling of [18F]CF3SO2NH4
[18F]Fluoride (3.0-4.0 GBq) was separated from 18O-enriched-water using a Chromafix PSHCO3 18F separation cartridge (45 mg, activated by slowly passing through 1 mL of H2O) and subsequently released with 900 μL (in 6×150 μL portions) of the K222/K2CO3 solution into a 5 mL V-vial containing a magnetic stir bar in the concentrator. The solution was dried with five cycles of azeotropic drying with anhydrous MeCN (5×200 μL) under a flow of N2 at 105° C. The dried [18F]KF/K222 residue was re-dissolved in anhydrous DMF (1000 μL). A solution of [18F]KF/K222 in DMF (20-30 MBq, 10-50 μL) was dispensed into a V-vial containing difluorocarbene reagent, amine-SO2 adduct and a magnetic stirrer bar. Anhydrous solvent (300 μL) was added via syringe before stirring at the specified temperature and time (See Table 1 to Table 4). The reaction was quenched with 10% EtOH in H2O (200 μL). An aliquot was removed for analysis by radioTLC and radioHPLC. Analysis was performed using the gradient given below with an analytical Synergi 4 μm Hydro-RP 80 A column, 150×4.6 mm at a flow rate 1 mL/min. Radio-TLC was performed on Merck Kiesegel 60 F254 plates, using MeCN:MeOH:H2O:AcOH 20:5:5:1 as eluent. Analysis was performed using a plastic scintillator/PMT detector. Radiochemical conversions are calculated from radioTLC and radioHPLC:
aRefer to Scheme 4.
bBased-on n = 2.
cBased-on n = 4.
aBased-on n = 2 unless otherwise stated.
bBased-on n = 4.
aContained about 20-70 μL of DMF as [18F]KF/K222 was dissolved in DMF and dispensed into reaction vials.
bBased-on n = 2 except for entry 5.
cBased-on n = 4.
dPDFA is suspened in a solution of NMM-SO2 in propylene carbonate and added to [18F]KF/K222 in about 50 μL of DMF.
aBased on n = 2.
b0.03 mmol of phosphine
c0.16 mmol of JohnPhos, 0.016 mmol of ClCF2CO2CH3, and 0.06 mmol of NMM-SO2 instead.
Mini Isolation Mode Screening
[18F]Fluoride (200-900 MBq) was separated from 18O-enriched-water using a Waters Sep-Pak light Accell Plus QMA cartridge (46 mg, activated with 2 mL water) and was subsequently released using a solution of Kryptofix-2.2.2 (15 mg) and K2CO3 (3 mg) in 1000 μL of MeCN/H2O, 4:1 into a 5 mL V-vial containing a magnetic stirrer bar in a concentrator.
The solution was dried with five cycles of azeotropic drying with anhydrous MeCN (5×200 μL) under a flow of N2 at 105° C.
For the transfer of reagents to [18F]fluoride (See Table 5 entry 4 to 6, Table 6 entry 2 to 5): anhydrous solvent (300 μL) was added to a 1.5 mL vial containing amine-SO2 adduct and difluorocarbene reagent before shaking to a fine suspension using a vortex mixer (˜10 seconds). The mixture was added to the dried [18F]KF/K222 residue in a V-vial via syringe.
For the transfer of [18F]fluoride to reagents (See Table S5 entry 1 to 3, Table S6 entry 1 and Table S9): anhydrous solvent (300 μL) was added to the dried [18F]KF/K222 residue and the mixture was heated at 100° C. for 2 min. The mixture was added to a V-vial containing amine-SO2 adduct and difluorocarbene reagent.
After stirring at the specified temperature and time, the reaction mixture was cooled, taken up in 2% formic acid in water (6 mL) and rinsed over a WAX cartridge (activated with 3 mL water and 3 mL 2% formic acid in water). The vial was then rinsed with 2 mL of EtOH before eluting over the WAX cartridge. The cartridge was then rinsed with EtOH (4 mL) before eluting [18F]CF3SO2NH4 into a 5 mL vial with 1% NH3 in EtOH (3 mL) and a stream of N2. An aliquot was removed to determine radiochemical purity by radioHPLC with an analytical Synergi 4 μm Hydro-RP 80 A column, 150×4.6 mm at a flow rate 1 mL/min. Activity was measured using a plastic scintillator/PMT detector. Radiochemical yields are calculated from activity and radiochemical purity:
Radiocemical Yield (%)=Radioc
emical Purity (%)×Final Activity (MBq)/Initial Activity (MBq)
aTransfer efficiency refers to the amount of [18F]KF/K222 that was transferred to the reaction vial by dissolving in DMF after azeotropic drying.
b0.2 mmol of NMM-SO2, 10 min and 100 .
c0.3 mmol of NMM-SO2, 20 min and 110
d0.16 mmol of PDFA and 0.06 mmol of NMM-SO2 were used.
efluoride was eluted with K222 only. QMA was preconditioned with Na2SO4 according to Mossine et al.8
aTransfer efficiency refers to the amount of [18F]KF/K222 that was transferred to the reaction vial by dissolving in propylene carbonate after azeotropic drying.
b0.2 mmol of NMM-SO2, 10 min and 100 .
c0.3 mmol of NMM-SO2, 20 min and 110
dPDFA was suspended in a solution of NMM-SO2.
e50 μL of DMF was added.
fSolvent comprised of 150 μL of DMF and 150 μL of propylene carbonate.
g0.16 mmol of PDFA and 0.06 mmol of NMM-SO2.
and 20 min of reaction time.
aBased-on n = 2.
bJohnPhos was added to the v-vial and the fluoride was dispensed into it, they were then azeotropically dried.
and 20 min of reaction time.
aPhosphine was added to the v-vial and the fluoride was dispensed into it, they were then azeotropically dried.
bRefer to scheme 6 for structure of phosphine.
aStandard conditions: 0.16 mmol of (o-toy1)3P, 0.16 mmol of ClCF2CO2CH3, 0.06 mmol of NMM-SO2, 300 μL of DMA, 110 and 20 min of reaction time. Phosphine was dissolved in DMA together with NMM-SO2. Gentle heating with a hair dryer was used.
b[18F] fluoride was eluted with a solution of dibenzo-24-crown-8 (8 mg), Cs2C2O4 (2.1 mg) and Cs2CO3 (100 μL of 2.5 mg/mL solution), 100 μL of water and 800 μL of MeCN. 900 μL of this solution was used in the elution.
Isolation Procedure of [18F] Ammonium Trifluoromethanesulflnate
[18F]Fluoride (5-10 GBq) was separated from 18O-enriched-water using a Waters Sep-Pak light Accell Plus QMA cartridge (46 mg, activated with 2 mL H2O) and was subsequently released using a solution of Kryptofix-2.2.2 (6.3 mg) and κ2CO3 (1 mg) in 1000 μL of MeCN/water, 4:1 into a 5 mL V-vial containing a magnetic stirrer bar in a concentrator. The solution was dried with five cycles of azeotropic drying with anhydrous MeCN (5×200 μL) under a flow of N2 at 105° C. A suspension of PDFA (57 mg, 0.16 mmol), and N-methylmorpholine-SO2 in Propylene Carbonate (300 μL) and DMF (50 μL) was then added and the reaction stirred at 125° C. (actual temperature is about 110° C.) for 20 minutes. After stirring at the specified temperature and time, the reaction mixture was cooled for 3 minutes, taken up in 2% formic acid in H2O (6 mL) and added to a WAX cartridge (activated with 3 mL H2O and 3 mL 2% formic acid in water). The vial was then rinsed with 2 mL of EtOH before eluting over the WAX cartridge. The WAX cartridge was then washed with EtOH (4 mL) before eluting [18F]CF3SO2NH4 into a 5 mL vial with 1% NH3 in EtOH (3 mL) and a stream of N2. The ethanol was then removed under a stream of N2 at 120° C. The vial was then cooled for 3 minutes before NH4CO2H (25 mM) was added, the vial shaken vigorously, and the solution loaded directly onto a 2 mL HPLC loop and injected onto a semi-Prep HPLC column (Synergi 4 μm Hydro-RP 250×10 mm) and eluted into a collection vial with 25 mM NH4HCO2 in water monitoring with UV (254 nm) and radioactive traces. The Molar Activity of [18F]NH4SO2CF3 was assessed by radio-HPLC, using an analytical Synergi 4 μm Hydro-RP 80 Å column, 150×4.6 mm eluted with 1% MCCN/99% 25 mM NH4HCO2 in H2O (isocratic 1 mL/min), monitoring with UV (220 nm) and radioactive traces.
Additional Notes for this Procedure:
It was found that even with minimal quantities of EtOH that the retention time of the product could drastically change and as such make purification challenging and less reliable.
Analysis of 5-[18F] Ammonium Trifluoromethanesulfonate ([18F]1)
General Procedure for the 18F-Trifluoromethylation of Peptides Using [18F]NH4SO2CF3
An aliquot of a solution of [18F]NH4SO2CF3 in 25 mM NH4HCO2H in water (20-300 μl, 10-100 MBq) was added to a v-vial which contained the substrate and the iron salt. A stock solution of 70% TBHP (aq) of the required concentration was prepared in the reaction solvent and an appropriate amount was taken up into a 1 mL syringe and added to the v-vial. The sealed vial was stirred at room temperature for 20 minutes.
Determination of Radiochemical Conversion: The reaction mixture was diluted with DMSO or 5% MeCN in water with 0.05% TFA and an aliquot was removed for analysis by and radio-HPLC to determine radiochemical yield. Radio-HPLC was performed on a Phenomenex Synergi Hydro RP 4 μm 80 Å 150×4.6 mm column, Agilent Zorbax 300 Extended C-18 4 μm 300 Å 150×4.6 mm column or Agilent Zorbax 300SB CN 4 μm 300 Å 150×4.6 mm column. Within the samples tested, all radioactive by-products are sufficiently soluble such that the radiochemical yield determined directly from HPLC and from isolation has no significant difference.
HPLC Eluent System for Peptides' Reactions
aWith 0.1% trifluoroacetic acid
aWith 0.1% trifluoroacetic acid
aWith 0.1% trifluoroacetic acid
aWith 0.1% trifluoroacetic acid
aWith 0.1% trifluoroacetic acid
aWith 0.1% trifluoroacetic acid
aWith 0.1% trifluoroacetic acid
aWith 0.1% trifluoroacetic acid
aWith 0.1% trifluoroacetic acid
aWith 0.1% trifluoroacetic acid
Isolation of Radiochemially Pure Products
Semi prep HPLC of crude reactions were performed with Phenomenex Onyx Monolithic Semi-PREP C18 100×10 mm.
aWith 0.1% trifluoroacetic acid
L-Tyr[ortho-CF218F]
Prepared following the general procedure using L-tyrosine, Fe(NO3)3.9H2O and 70% TBHP (aq). The amount and condition used are specified in Table 22. Radiochemical conversion was determined by HPLC (Eluent H, Table 18, at 40° C. with the Phenomenex Synergi Hydro RP 4 μm 80 Å 150×4.6 mm column). The HPLC overlay of crude radio-trace of L-tyrosine reaction with [18F]NH4SO2CF3 and UV trace of authentic reference for both the ortho and meta-CF3 products are provided in
anonahydrate was used.
bAF = Ammonium Formate.
aDetermined from relative area of all radiopeaks in HPLC.
bHPLC was performed with Zorbax 300 Extended C-18 4 μm 300 Å 150 × 4.6 mm column instead.
L-Trp[W-2-CF218F]
Prepared following the general procedure using L-tryptophan (0.03 mmol, 5.4 mg), FeCl3 (0.06 mmol, 6.1 mg) and 70% TBHP in water (0.12 mmol, 16.7 μL), the amount of solvent is specified in Table 25. Radiochemical conversion was determined by radio-HPLC (Refer to Table 26 and Table 27). HPLC overlay spectra for the products are provided in
aDetermined from relative area of all radiopeaks in HPLC.
bRefer to Table 13.
cRefer to Table 12.
aDetermined from relative area of all radiopeaks in HPLC.
bRefer to Table 13.
cRefer to Table 12.
Isolation with an OASIS HLB plus: The HLB plus cartridge is activated with 6 mL of EtOH, blown dry then washed with 6 mL of H2O. The crude reaction mixture (4.96 MBq) was diluted with 4 mL of H2O and taken up into a syringe. The crude mixture was eluted slowy through the HLB plus cartridge. The vial with the crude reaction mixture was washed with 1 mL of H2O and the washing was eluted through the same HLB plus. 4 mL of H2O was eluted through the HLB plus cartridge. 2 mL of EtOH was used to elute the product from the HLB cartridge. The eluted activity was measured to be 2.12 MBq. HPLC analysis was performed on the eluted mixture, the radiochemical purity of the product is 73.67%. The decay uncorrected radiochemical yield is 31.5%.
TyrTrp[W-2-CF218F]
Prepared following the general procedure using H-Tyr-Trp-OH (0.015 mmol), Fe(NO3)3.9H2O (0.03 mmol) and 70% TBHP (0.06 mmol), except in the case of Table S28 Entry 1 where substrates and reagents are double and Entry 6 where FeCl3 was used instead of Fe(NO3)3□9H2O. The reaction mixture was stirred at 40° C. for 20 min. Radiochemical conversion was determined by radio-HPLC (Eluent A, Table S11, at 40° C. with Phenomenex Synergi Hydro RP 4 μm 80 Å 150×4.6 mm column unless otherwise indicated). HPLC overlay spectra for the crude reaction for Table 38, entry 2, and for the separated reaction products are provided in
aDetermined from relative area of all radiopeaks in HPLC.
bHPLC was performed with Zorbax 300 Extended C-18 4 μm 300 Å 150 × 4.6 mm column instead.
aDetermined from relative area of all radiopeaks in HPLC.
Isolation with an OASIS HLB plus: The HLB plus cartridge is activated with 6 mL of MeOH, blown dry then washed with 6 mL of H2O. The crude reaction mixture (5.86 MBq) was diluted with 4 mL of H2O and taken up into a syringe. The crude mixture was eluted slowly through the HLB plus cartridge. The vial with the crude reaction mixture was washed with 1 mL of H2O and the washing was eluted through the same HLB plus. 4 mL of H2O was eluted through the HLB plus cartridge. 2 mL of EtOH was used to elute the product from the HLB cartridge. The eluted activity was measured to be 4.4 MBq. HPLC analysis was performed on the eluted mixture, the radiochemical purity of the product is 53.95%. The decay uncorrected radiochemical yield (RCY) is 27.9%. The RCY determined directly from HPLC without isolation of the crude reaction mixture is 28.3%.
Trp[W-2-CF218F]Tyr
Prepared following the general procedure using H-Trp-Tyr-OH (0.015 mmol, 5.5 mg), Fe(NO3)3.9H2O (0.03 mmol, 12.1 mg) and 70% TBHP in water (0.06 mmol, 8.2 μL), the amount of solvent is specified in Table 31. Radiochemical conversion was determined by radio-HPLC (Refer to Table 32, Table 33 and Table 34). HPLC overlay spectra for the products are provided in
aDetermined from relative area of all radiopeaks in HPLC.
bTable 11.
cRefer to Table 16.
aDetermined from relative area of all radiopeaks in HPLC.
bTable 11.
cRefer to Table 16.
aDetermined from relative area of all radiopeaks in HPLC.
bTable 11.
cRefer to Table 16.
PheTyr[ortho-CF218F]
Prepared following the general procedure using H-Phe-Try-OH (0.03 mmol, 9.9 mg), Fe(NO3)3□9H2O (0.06 mmol, 24.2 mg) and 70% TBHP in water (0.12 mmol, 16.7 μL), the amount of solvent is specified in Table 35. Radiochemical conversion was determined by radio-HPLC (Refer to Table 36 and Table 37). HPLC overlay spectra for the products are provided in
aDetermined from relative area of all radiopeaks in HPLC.
bRefer to Table 11
cRefer to Table 13.
dRefer to Table 14.eRefer to Table 19.
aDetermined from relative area of all radiopeaks in HPLC.
bRefer to Table 11.
cRefer to Table 13.
dRefer to Table 14.
eRefer to Table 19.
PheTrp[W-2-CF218F]
Prepared following the general procedure using H-Phe-Trp-OH (0.015 mmol, 5.3 mg), Fe(NO3)3.9H2O (0.03 mmol, 12.1 mg) and 70% TBHP in water (0.06 mmol, 8.2 μL), the amount of solvent is specified in Table 38. Radiochemical conversion was determined by radio-HPLC (Refer to Table 39 and Table 40). HPLC overlay spectra for the products are provided in
aDetermined from relative area of all radiopeaks in HPLC.
bRefer to Table S11.
cRefer to Table S13.
aDetermined from relative area of all radiopeaks in HPLC.
bRefer to Table S11.
cRefer to Table S13.
MetTrp[W-2-CF218F]
Prepared following the general procedure using H-Met-Trp-OH (0.03 mmol, 9.4 mg), Fe(NO3)3□9H2O (0.06 mmol, 24.2 mg) and 70% TBHP in water (0.06 mmol, 8.3 μL), the amount of solvent is specified in Table 41. Radiochemical conversion was determined by radio-HPLC (Refer to Table 42 and Table 43). HPLC overlay spectra for the products are provided in
aDetermined from relative area of all radiopeaks in HPLC.
bRefer to Table 11
aDetermined from relative area of all radiopeaks in HPLC.
bRefer to Table 11
MetTyr[ortho-CF218F]
Prepared following the general procedure using H-Met-Tyr-OH (0.03 mmol, 9.4 mg), Fe(NO3)3□9H2O (0.06 mmol, 24.2 mg) and 70% TBHP in water (0.06 mmol, 8.3 μL), the amount of solvent is specified in Table 44. Radiochemical conversion was determined by radio-HPLC (Refer to Table 45, Table 46 and Table 47). HPLC overlay spectra for the products are provided in
aDetermined from relative area of all radiopeaks in HPLC.
bRefer to Table S11
aDetermined from relative area of all radiopeaks in HPLC.
bRefer to Table S11
aDetermined from relative area of all radiopeaks in HPLC.
bRefer to Table S11
Tyr[ortho-CF218F]His
Prepared following the general procedure using H-Tyr-His-OH (0.03 mmol, 9.6 mg), Fe(NO3)3.9H2O (0.06 mmol, 24.2 mg) and 70% TBHP in water (0.12 mmol, 16.6 μL), the amount of solvent is specified in Table 48. Radiochemical conversion was determined by radio-HPLC (Refer to Table 49 and Table 50). HPLC overlay spectra for the products are provided in
aDetermined from relative area of all radiopeaks in HPLC.
bRefer to Table 11
aDetermined from relative area of all radiopeaks in HPLC.
bRefer to Table 11
HisTrp[W-2-CF218F]
Prepared following the general procedure using H-His-Trp-OH (0.015 mmol, 5.1 mg), Fe(NO3)3.9H2O (0.03 mmol, 12.1 mg) and 70% TBHP in water (0.06 mmol, 8.2 μL), the amount of solvent is specified in Table 51. Radiochemical conversion was determined by radio-HPLC (Refer to Table 52, Table 53 and Table 54). HPLC overlay spectra for the products are provided in
aDetermined from relative area of all radiopeaks in HPLC.
bRefer to Table S11
aDetermined from relative area of all radiopeaks in HPLC.
bRefer to Table S11
Glu-Trp[W-2-CF218F]
Prepared following the general procedure using H-Glu-Trp-OH (0.03 mmol, 10.0 mg), FeCl3 (0.06 mmol, 9.7 mg) and 70% TBHP in water (0.06 mmol, 8.2 μL), the amount of solvent is specified in Table 55. Radiochemical conversion was determined by radio-HPLC (Refer to Table 56 and Table 57). HPLC overlay spectra for the products are provided in
aAdditonal 25 mM NH4HCO2 added beside those dispensed with the [18F]NH4HCO2.
aDetermined from relative area of all radiopeaks in HPLC.
bRefer to Table S11
aDetermined from relative area of all radiopeaks in HPLC.
bRefer to Table S11
Isolation Results
aDue to the limitation of the HPLC loop only 100 μL can be injected each time. This limitation is operational and can be circumvented if required. bRadiochemical Yield = Isolated Activity/Injected Activity × 100 and no decay correction was applied. The Radiochemical purity of the isolated product is >99%. See Figure S41.
Angiotensin (1-7)[Y-ortho-CF218F]
Prepared following the general procedure using angiotensin(1-7) (0.0092 mmol, 12 mg), Fe(NO3)3.9H2O (0.06 mmol, 24.2 mg) and 70% TBHP in water (0.06 mmol, 8.2 μL), the amount of solvent is specified in Table 59. Radiochemical conversion was determined by radio-HPLC (Refer to Table 60 and Table 61). HPLC overlay spectra for the products are provided in
a Additonal 25mM NH4HCO2 added beside those dispensed with the [18F]NH4HCO2.
aDetermined from relative area of all radiopeaks in HPLC.
bRefer to Table 20.
aDetermined from relative area of all radiopeaks in HPLC.
bRefer to Table 20.
Melittin[W-2-CF218F]
Prepared following the general procedure using Melittin (0.00074 mmol, 2.0 mg), FeCl3 (0.02 mmol, 3.2 mg) and 70% TBHP in water (0.02 mmol, 2.8 μL), the amount of solvent is specified in Table 59. Radiochemical conversion was determined by radio-HPLC (Refer to Table 63). HPLC overlay spectra for the product is provided in
aAdditonal 25 mM NH4HCO2 added beside those dispensed with the [18F]NH4HC02.
aDetermined from relative area of all radiopeaks in HPLC.
bRefer to Table S16.
Somatostatin-14[W-2-CF218F]
Prepared following the general procedure using Somatostatin-14 (0.0061 mmol, 10 mg), FeCl3 (0.08 mmol, 13.0 mg) and 70% TBHP in water (0.08 mmol, 11 μL), the amount of solvent is specified in Table 64. Radiochemical conversion was determined by radio-HPLC (Refer to Table 65). HPLC overlay spectra for the products are provided in
aDetermined from relative area of all radiopeaks in HPLC.
bRefer to Table 14.
Isolation Results
aDue to the limitation of the HPLC loop only 100 μL can be injected each time. This limitation is operational and can be circumvented if required.
bRadiochemical Yield = Isolated Activity/Injected Activity × 100 and no decay correction was applied. The Radiochemical purity of the isolated product is >99%. See FIG. 36.
Endomorphin 1[W-2-CF21F]
Prepared following the general procedure using Endomorphin 1 (0.004 mmol, 3 mg), FeCl3 (0.02 mmol, 3.2 mg) and 70% TBHP in water (0.02 mmol, 2.8 μL), the amount of solvent is specified in Table 67. Radiochemical conversion was determined by radio-HPLC (Refer to Table 68 and Table 69). HPLC overlay spectra for the products are provided in
aAdditonal 25 mM NH4HCO2 added beside those dispensed with the [18F]NH4HCO2.
aDetermined from relative area of all radiopeaks in HPLC.
bRefer to Table 16.
aDetermined from relative area of all radiopeaks in HPLC.
bRefer to Table 16.
Isolation Results
aDue to the limitation of the HPLC loop only 100 μL can be injected each time. This limitation is operational and can be circumvented if required.
bRadiochemical Yield = Isolated Activity/Injected Activity × 100 and no decay correction was applied. The Radiochemical purity of the isolated product is >99%. See FIG. 39.
Insulin
Prepared following the general procedure using recombinant human insulin (0.0052 mmol, 30 mg), Fe(NO3)3.9H2O (0.03 mmol, 12.1 mg) and 70% TBHP in water (0.06 mmol, 8.2 μL), the amount of solvent is specified in Table 71. Radiochemical conversion was determined by radio-HPLC (Refer to Table 72). HPLC overlay spectra for the products are provided in
aAdditonal 25 mM NH4HCO2 added beside those dispensed with the [18F]NH4HCO2.
aDetermined from relative area of all radiopeaks in HPLC.
bRefer to Table 16.
cRefer to Table 17.
dRefer to Table 14.
To cyclo(-Arg-Gly-Asp-D-Tyr-Lys) (5 mg, 0.008 mmol) and iron(III) chloride hexahydrate (8.8 mg, 0.032 mmol) in a 3 mL V-vial was added [18F]NH4SO2CF3 in aqueous NH4HCO2 solution (50-400 MBq) followed by tert-butyl hydroperoxide solution (8 μL, 0.06 mmol, 70% in H2O) in DMSO to give a total reaction solvent of 75% DMSO in aqueous NH4HCO2 (200 μL) before stirring at 40° C. for 20 mins. An aliquot was removed for analysis by radio-HPLC to give a RCC.
Reverse phase HPLC details: 16% MecN in 84% NH4HCO2 25 mM on a Phenomenex Synergi™ 4 μm Hydro RP 80 Å 150×4.6 mm column.
RCC (radiochemical conversion): 33%±15% (n=3, based on radio-HPLC analysis of the crude reaction, shown in
1H and 19F NMR, and TOF mass spectrometry results for the reference compound (19F-trifluoromethyl functionalised cyclo(-Arg-Gly-Asp-D-Tyr-Lys)) are shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 1815013.6 | Sep 2018 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2019/052577 | 9/13/2019 | WO | 00 |
