Pyridinium, quinolinium, acridinium, pyrylium, chromenylium or xanthylizum reactive desorption and/or laser ablation ionization matrices and use thereof

Abstract

The present disclosure concerns use of compounds of formula I, or salts thereof, as reactive matrices for desorption and laser ablation ionization spectrometry. The disclosure further concerns compounds of formula II, or salts thereof, and use of compounds of formula II or III, or salts thereof. ##STR00001##

Claims

1. A compound of formula II or a salt thereof: ##STR00051## wherein: ##STR00052## is selected from the group consisting of ##STR00053## —Ar.sup.1 is optionally substituted and is selected from the group consisting of biphenyl, terphenyl, C.sub.10-C.sub.30 polycyclic aromatic hydrocarbon and C.sub.6-C.sub.30 heteroaryl analogue of a polycyclic aromatic hydrocarbon; -L-is selected from a bond, —(CH.sub.2).sub.m— or any π-conjugating linker moiety selected from the group consisting of —O—, —S—, —NH—, ##STR00054## -G.sup.1 is selected from —H, -Me or —Ar.sup.2; —R is a C.sub.1-C.sub.15 alkyl group optionally labelled with one or more D, T, or .sup.13C atoms; -G.sup.2 is selected from the group consisting of —F, —Cl, —Br, —I and —(CH.sub.2).sub.n—Z—NH—NH.sub.2; m and n are each independently from 0 to 15; —Z— is selected from a bond, —CH.sub.2— or —C(O)—; and Ar.sup.2 is optionally substituted and is selected from the group consisting of phenyl, biphenyl, terphenyl, C.sub.10-C.sub.30 polycyclic aromatic hydrocarbon, and C.sub.4-C.sub.30 mono- or polycyclic heteroaryl.

2. Compound according to claim 1, wherein the compound of formula II has a formula IIa or IIb, ##STR00055## and wherein ##STR00056## is selected from the group consisting of ##STR00057##

3. Compound according to claim 1, wherein each of Ar.sup.1 and Ar.sup.2 is optionally independently substituted with one or more substituents selected from the group consisting of -D, -T, —F, —Cl, —Br, —I, —NO.sub.2, —CN, —R″′, —OR″′, —OC(O)R″′, —SR″′, —S(O)R″′, —S(O)(O)R″′ and —NR″′R″″, wherein —R″′ and —R″″ are each independently selected from phenyl, tolyl and C.sub.1-C.sub.15 alkyl.

4. Compound according to claim 1, wherein Ar.sup.1 is optionally substituted with one or more -Ph, —Cl or —Br groups and is selected from the group consisting of biphenyl, terphenyl, naphthalene, anthracene, phenanthrene, and pyrene.

5. Compound according to claim 1, wherein-G.sup.1 is —H.

6. Compound according to claim 1, wherein the compound of formula II, IIa or IIb is ##STR00058## wherein —R is optionally labelled with one or more deuterium, tritium, or carbon-13 atoms and is selected from the group consisting of methyl, ethyl, n-propyl and n-butyl; and -G.sup.2 is selected from the group consisting of —F, —Cl, —Br and —I.

7. Compound according to claim 1, wherein ##STR00059## Z is C═O, and wherein n is from 1 to 3.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) For a fuller understanding of the present invention and further objects and advantages of it, the detailed description set out below should be read together with the accompanying drawings, in which the same reference notations denote similar items in the various diagrams, and in which:

(2) FIG. 1a schematically illustrates derivatization of a nucleophile using a 2-substituted N-alkylpyridinium-based reactive matrix

(3) FIG. 1b schematically illustrates derivatization of a carbonyl-containing compound using a hydrazine-based reactive matrix

(4) FIG. 1c schematically illustrates derivatization of a primary amine using a pyrylium-based reactive matrix

(5) FIG. 2a schematically illustrates a general synthetic method for producing 2-substituted N-alkylpyridinium-based reactive matrices

(6) FIG. 2b schematically illustrates a range of 2-substituted N-alkylpyridinium compounds that may be produced by the general synthetic method illustrated in FIG. 2a

(7) FIG. 3 schematically illustrates a general synthetic method for producing N-substituted pyridine reactive matrices having a hydrazine reactive moiety

(8) FIG. 4 is a MALDI mass spectrum illustrating detection of a hydrazone derivative of 11-deoxycorticostereone without addition of any additional assisting matrix

(9) FIG. 5 is a series of images (a)-(f) illustrating the MALDI-MSI analysis of fluvoxamine in mouse brain tissue sections

DETAILED DESCRIPTION

(10) The reactive matrices of the invention may facilitate the mass spectrometric analysis, e.g. MALDI-MS(I) and DESI-MS(I) analysis, of a broad range of compounds including pharmaceuticals and their metabolites, endogenous neurotransmitters, amino acids, metabolites, steroids, toxins as well as environmental pollutants, food additives, food ingredients and any other small molecules containing primary and secondary amines, phenolic hydroxide and carbonyl functional groups. The reactive matrices of the invention are important tools for rapid monitoring/molecular imaging of above-mentioned compounds in the following field for example: biology in general, medicine in general, neuroscience, cancer research, drug development, toxicology, environmental science and food chemistry.

(11) The reactive matrices are applicable to a broad range of mass spectrometric (MS) and mass spectrometric imaging (MSI) techniques that utilize desorption and/or laser ablation. Such techniques include, but are not limited to:

(12) Matrix-assisted laser desorption/ionization (MALDI-MS, MALDI-MSI);

(13) Desorption electrospray ionization (DESI-MS, DESI-MSI);

(14) Matrix-assisted laser desorption electrospray ionization (MALDESI-MS, MALDESI-MSI);

(15) Nanospray desorption electrospray ionization (nano-DESI-MS, nano-DESI-MSI);

(16) Secondary ion mass spectrometry (SIMS, SIMS-MSI);

(17) Matrix-enhanced secondary ion mass spectrometry (ME-SIMS, ME-SIMS-MSI); and

(18) Laser ablation electrospray ionization (LAESI-MS, LAESI-MSI).

(19) The reactive matrices may also be utilizable in other forms of spectroscopy or mass spectrometry. For example, they may be used to label molecules with a chromophore prior to UV-spectroscopy or UV-imaging. They may also be used to provide a permanent charge (“charge-tagging”) to molecules prior to mass spectrometry using electrospray ionization (ESI), and thus may be utilized in liquid extraction surface analysis (LESA) comprising either ESI or MALDI.

(20) By reactive matrix it is meant a compound that may react with one or more target analyte molecules and facilitate the mass spectroscopic analysis of the target molecule(s). In some cases, use of the reactive matrix may completely avoid the need to use a further matrix to assist ionization. However, in some cases the reactive matrices may also be used together with a conventional non-reactive matrix in order to improve detection of the target molecule(s).

(21) The reactive matrices disclosed herein incorporate at least two functional domains: a highly reactive domain that facilitates covalent analyte charge-tagging by derivatization; and a conjugated chromophore domain to promote laser-assisted desorption and/or ablation. However, in some cases wherein the reactive domain is part of a polycyclic ring system the reactive domain and conjugated chromophore domain may be integrated in one and the same ring system. The reactive matrices may also incorporate an identification domain; that is to say a suitable isotope or element having a distinctive isotope pattern and mass defect in order to facilitate mass-spectrometric identification of compounds that are derivatised by the reactive matrix.

(22) Reactive Domain

(23) The reactive domain of the reactive matrices may be an N-alkylpyridinium moiety having a suitable leaving group, an N-substituted pyridine having a hydrazine moiety, or a pyrylium moiety. All of these reactive domains have the advantage of providing a permanent positive charge to the analyte after derivatization, due to the presence after derivatization of an N-substituted pyridine moiety. This vastly increases the sensitivity of the analyte for positive ion mass spectrometry, especially for analytes that are otherwise difficult to protonate.

(24) A reactive N-alkylpyridinium moiety having a suitable leaving group may selectively react with molecules comprising any one of a variety of common nucleophilic functional groups, including phenolic hydroxyl groups and primary and secondary amines. A reaction scheme demonstrating such derivatization reactions is shown in FIG. 1a. The reaction takes place without any need for extended incubation times. The leaving group (G.sup.2 group) of the N-alkylpyridine may be any suitable leaving group for nucleophilic aromatic substitution. Such leaving groups include —F, —Cl, —Br, —I, —NO.sub.2, —CN, —OR′, —OC(O)R′, —SR′, —S(O)R′, —S(O)(O)R′ and —NR′R″, wherein R′ and R″ are each independently phenyl or C.sub.1-C.sub.15 alkyl groups. The leaving group may suitably be positioned at the 2- or 4-positions of the pyridine ring, which are expected to be the most reactive positions for nucleophilic aromatic substitution (S.sub.NAr). However, arrangement of the leaving group at the 3-position has been demonstrated to also provide sufficient reactivity, at least in some circumstances. The N-alkyl group is described below.

(25) An N-substituted pyridine having a reactive hydrazine moiety may selectively react with any analytes having a carbonyl functionality, as shown in FIG. 1b. The reaction proceeds rapidly. The hydrazine moiety is bound by a linker to either the N-position or another position of the pyridine ring, such as the 2-position or 4-position. The linker may suitably be an alkyl chain, optionally comprising a carbonyl group closest to the hydrazine moiety. Alternatively, the linker may be a direct bond between the N-substituted pyridine and a nitrogen of the hydrazine moiety. If the hydrazine moiety is bound (via the linker) to a carbon of the pyridine ring, the N-position of the pyridine ring is alkylated in order to provide a permanently charged N-alkyl pyridine.

(26) The N-alkyl group of the pyridine reactive domains may be any suitable group, such as a C.sub.1-C.sub.15 alkyl group. The N-alkyl group may be isotope-enriched, for example by having an abundance of .sup.2H, .sup.3H, .sup.13C or .sup.14C at a specific position. This may facilitate identification of the derivatized analytes by mass spectrometry. The N-alkyl group may be a chiral group allowing for stereoisomeric discrimination between analytes. Where the reactive domain is a 2-substituted N-alkylpyridinium, the N-alkyl group is preferably non-bulky in order to enable the nucleophilic substitution reaction at the 2-position of the pyridine ring. For example, the N-alkyl group may be a methyl group.

(27) A pyrylium reactive moiety may selectively react with analytes having a primary amine functionality, resulting in an N-alkyl pyridine derivative as shown in FIG. 1c. This reaction may require substantial incubation times, depending on the exact nature of the pyrylium moiety and analyte.

(28) Conjugated Chromophore Domain

(29) The conjugated chromophore domain of the reactive matrix comprises an Ar.sup.1 group selected from the group consisting of phenyl, biphenyl, terphenyl, C.sub.10-C.sub.30 polycyclic aromatic hydrocarbon and C.sub.4-C.sub.30 mono- or polycyclic heteroaryl. By polycyclic aromatic hydrocarbon it is meant a carbocyclic aromatic ring system comprising two or more fused rings. Suitable polycyclic aromatic hydrocarbons are for example listed as structures numbers 4-521 in NIST Special Publication 922 (revised June 2011) “Polycyclic Aromatic Hydrocarbon Structure Index”, which is hereby incorporated by reference. The polycyclic aromatic hydrocarbon may for example be selected from the group consisting of naphthalene, fluorine, anthracene, phenanthrene, pyrene, tetracene, chrysene, perylene, corannulene, coronene and pyranthrene. By C.sub.4-C.sub.30 mono- or polycyclic heteroaryl, it is meant any heteroaryl ring system from 5-membered rings and upwards (i.e. furan, thiophene, pyrrole), including any heteroaryl analogues of the polyaromatic hydrocarbons as defined above. The heteroaryl rings may comprise any combination of carbon and heteroatoms, including but not limited to oxygen, sulfur and nitrogen atoms. The conjugated chromophore domain preferably exhibits strong optical absorption at the wavelength of the pulsed laser (typically 337 nm for nitrogen or 355 nm for Nd:YAG laser). For example, having a conjugated chromophore domain comprising an anthracene or substituted anthracene moiety provides reactive matrices having a suitable absorptivity, without having an excessive molar mass.

(30) The Ar.sup.1 group may be provided with one or more substituents in order to tailor the properties of the reactive matrix. Suitable substituents may for example include -D, -T, —F, —Cl, —Br, —I, —NO.sub.2, —CN, —R″′, —OR″′, —OC(O)R″′, —SR″′, —S(O)R″′, —S(O)(O)R″′ and —NR″′R″″, wherein —R″′ and —R″″ are each independently selected from phenyl, tolyl and C.sub.1-C.sub.15 alkyl.

(31) By “conjugated chromophore domain” it is meant that the chromophore comprises a conjugated π-system, and not necessarily that the chromophore is conjugated with the reactive domain. However, in order to provide suitable absorptivity the conjugated system may extend into the pyridine/pyrylium ring of the derivatized analyte. This is particularly preferable in cases where the Ar.sup.1 group by itself does not have a sufficiently extended π-system in order to absorb at the wavelengths mentioned above. This is achieved by connecting the Ar.sup.1 group to the pyridine or pyrylium moiety using a π-conjugating linker moiety. By π-conjugating linker moiety it is meant a moiety providing an uninterrupted π-conjugation path between the Ar.sup.1 group and the pyridine/pyrylium moiety, i.e. a direct bond between the Ar.sup.1 group and the pyridine/pyrylium moiety, or a moiety comprising only sp and/or sp.sup.2 hybridized atoms in a path directly connecting the Ar.sup.1 group to the pyridine/pyrylium moiety. Such a moiety may as noted above be a direct bond between the Ar.sup.1 group and the pyridine/pyrylium moiety, or it may be a moiety selected from the group consisting of —O—, —S—, —NH—,

(32) ##STR00024##

(33) The π-conjugating linker moiety may comprise pendant groups comprising sp.sup.3 hybridized atoms, wherein by pendant groups it is meant groups not directly incorporated in the conjugation path between the Ar.sup.1 group and the pyridine/pyrylium moiety. For example, the π-conjugating linker moiety may comprise chiral pendant groups in order to facilitate stereoisomeric discrimination between analytes. The π-conjugating linker moiety may also allow for axial chirality (atropisomerism) due to restricted rotation of the Ar.sup.1 group relative to the pyridine/pyrylium moiety. This may especially be the case where the π-conjugating linker moiety is a direct bond. However, if the Ar.sup.1 group by itself has suitable absorption, it is not necessarily advantageous to extend the conjugated system into the pyridine/pyrylium ring, and in such cases a non-conjugating linker, such as an alkyl chain or any other chain comprising sp.sup.3 hybridized carbon atoms, may be used to connect the pyridine/pyrylium ring to the Ar.sup.1 group.

(34) A suitable G.sup.1 group may be chosen to tailor the chromophore and reactive properties of the reactive matrix. G.sup.1 may be selected from —H, -Me or—Ar.sup.2, wherein —Ar.sup.2 is optionally substituted and is selected from the group consisting of phenyl, biphenyl, terphenyl, C.sub.10-C.sub.30 polycyclic aromatic hydrocarbon, and C.sub.4-C.sub.30 mono- or polycyclic heteroaryl. If G.sup.1 is an aryl (Ar.sup.2) group, this may further extends the π-conjugation into the G.sup.1 group, thus altering the absorptivity of the reactive matrix. The reactive and chromophore properties of the reactive matrix may be further tuned by providing the —Ar.sup.2 group with one or more substituents when G.sup.1 is an Ar.sup.2 group. Suitable substituents may include -D, -T, —F, —Cl, —Br, —I, —NO.sub.2, —CN, —R′, —OR″′, —OC(O)R″′, —SR″′, —S(O)R″′, —S(O)(O)R″′ and —NR″′R″″, wherein —R″′ and —R″″ are each independently selected from phenyl, tolyl and C.sub.1-C.sub.15 alkyl.

(35) Where the reactive domain is a pyrylium moiety, a suitable G.sup.3 group may be chosen to tailor the chromophore and reactive properties of the reactive matrix. The G.sup.3 group may independently be selected from the same substituents as for the G.sup.1, as described above.

(36) Integrated Reactive Domain and Conjugated Chromophore Domain

(37) In some cases wherein the reactive domain is part of a polycyclic ring system, a separate conjugated chromophore domain may not be required due to the reactive domain by itself having sufficiently good chromophore properties, i.e. the reactive domain and conjugated chromophore domain may be integrated into one and the same ring system. This for example may be the case where the reactive domain is a fused N-alkylpyridinium moiety, such as an N-alkylacridine moiety or an N-alkylquinoline moiety. Such integrated systems may provide improved atom economy.

(38) Identification Domain

(39) The reactive matrices may be provided with an identification domain that facilitates mass spectrometric identification of analytes charge-tagged with the reactive matrix. This may for example be achieved by providing a chlorine or bromine substituent on the reactive matrix, since these atoms provide a distinctive mass spectroscopic isotope pattern and mass defects.

(40) Alternatively, the reactive matrix may be isotope enriched by substitution with deuterium, tritium or an alkyl or aryl group enriched with a carbon isotope such as .sup.13C or .sup.14C. By labelled, isotope-enriched or substituted with a particular isotope, it is meant that the occurrence of a particular isotope (e.g. .sup.2H, .sup.3H, .sup.13C or .sup.14C) at a certain position in the molecule vastly exceeds the naturally occurring abundance at that position. For example, the ratio of the “identification” isotope (e.g. .sup.2H, .sup.3H, .sup.13C or .sup.14C) to the most abundant naturally occurring isotope (e.g. 1H or .sup.12C) at the relevant position may be at least 1:10, such as at least 1:1 or at least 10:1.

(41) The reactive matrices possess a permanent positive charge and thus may be prepared as salts with one or more anionic counterions. Such salts may for example comprise one or more anions selected from the non-exhaustive group consisting of chloride, bromide, iodide, toslylate, mesylate, tetrafluoroborate, perfluorobutanesulfonate, benzenesulfonate, hexafluorophosphate and triflate.

(42) Use of the Reactive Matrices in Desorption and/or Laser Ablation Ionization Mass Spectrometry

(43) When preparing a sample for mass spectrometric analysis or imaging, the reactive matrix substances in most cases do not need any incubation with the sample. The reaction occurs at the same time when the reagent is added/spotted/sprayed to/over the sample without any need for cooling or heating. Depending on the nature of the reactive domain, the reactive matrices target substances with primary amine, secondary amine, phenolic hydroxide, aldehyde or ketone functional groups. All of the reactive matrices carry a permanent positive charge after reacting with the functional group of the analyte. This allows for high sensitivity detection and analysis of the derivatized analytes.

EXAMPLES

Example 1: Synthesis of 2-Substituted N-alkylpyridinium Reactive Matrices

(44) 2-substituted N-alkylpyridinium reactive matrices are synthesised by a modular synthetic route as schematically illustrated in FIG. 2a. The two domains (chromophore and reactive domain) are fused using a Suzuki-Miyara cross-coupling reaction in step A. Thus, treatment of the appropriate 2-substituted bromopyridine derivative with a range of aryl boronic acids under palladium-catalysis afforded the precursor compounds in moderate to excellent yield. These precursors may in an optional step B be regioselectively brominated with N-bromosuccinimide to introduce an identification domain. The precursors are then subsequently alkylated in a step C under thermal conditions to afford afforded the desired reactive matrix. A selection of reactive matrices produced by this method are shown in FIG. 2b. Notably the final compounds were readily isolated by simple filtration, which negated the need for a potentially problematic purification step. The compounds were found to be stable upon storage under vacuum for months, however they degrade slowly when stored in solution.

General Procedure A for Synthesis of 2-fluoro pyridines Exemplified by 2-fluoro-4-phenylpyridine

(45) All reagents were purchased at the highest commercial quality and used without further purification. Yields refer to isolated, homogenous and spectroscopically pure material, unless otherwise stated. Crude reaction mixtures were purified by silica gel chromatography (E. Merck silica gel, particle size 0.043-0.063 mm). Thin layer chromatography was carried out using E. Merck silica plates (60F-254) with UV light (254 nm) as the visualization agent. .sup.1H NMR spectra were recorded at 400 MHz and .sup.13C{.sup.1H} NMRspectra at 100 MHz. The chemical shifts for .sup.1H NMR and .sup.13C{.sup.1H} NMR spectra were referenced to tetramethylsilane via residual solvent signals (.sup.1H, CDCl.sub.3 at 7.26 ppm, Acetone-d.sub.6 at 2.05 ppm, CD.sub.3CN at 1.95 ppm; .sup.13C, CDCl.sub.3 at 77.16 ppm, Acetone-d.sub.6 at 29.9 ppm, CD.sub.3CN at 1.39 ppm). LC/MS analysis was performed on an instrument equipped with a C18 column (50×3.0 mm, particle size 2.6 μm, pore size 100 Å) an electrospray ionization source and a single quadrupole detector. Accurate mass values were determined on a mass spectrometer equipped with an electrospray or MALDI ion source and TOF or FTICR analyzer.

(46) To a stirred solution of 4-bromo-2-fluoropyridine (528 mg, 3 mmol), phenylboronic acid (399 mg, 3.3 mmol), Pd(PPh.sub.3).sub.4 (69 mg, 0.06 mmol) in toluene/ethanol (4:1, 25 ml) was added a solution of Na.sub.2CO.sub.3 (1.27 g, 12 mmol) in water (10 ml). The resulting yellow solution was stirred at 80° C. for 2 hours, during which time the solution turned black. After cooling to ambient temperature the reaction mixture was extracted with EtOAc (3×50 ml). The combined organic layers were the washed with brine (50 ml), dried over MgSO.sub.4, filtered and concentrated in vacuo. The crude product was purified by hot filtration from MeOH followed by silica gel chromatography (5% EtOAc in n-pentane) to the title compound as a light yellow solid (490 mg, 2.8 mmol, 94%).

Optional General Procedure B for the Bromination of 2-fluoropyridines Exemplified by 4-(10-Bromoanthracen-9-yl)-2-fluoropyridine

(47) To a stirred solution of 4-(anthracen-9-yl)-2-fluoropyridine (715 mg, 2.75 mmol) in CHCl.sub.3 (35 ml) was added N-bromosuccinimide (1.08 g, 6.07 mmol) and the resulting solution was refluxed for 48 h. After cooling to room temperature, the mixture diluted with water (25 ml) and extracted with CH.sub.2Cl.sub.2 (3×25 ml). The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. Purification by flash column chromatography (SiO.sub.2, EtOAc:toluene:pentane 5:15:80 (v/v/v)) provided the title compound as a yellow solid (784 mg, 85%).

General Procedure C for the Preparation of N-alkyl arylfluoropyridium salts Exemplified by 2-fluoro-1-methyl-4-phenylpyridin-1-ium iodide

(48) 2-fluoro-4-phenylpyridine (200 mg, 1.15 mmol) was dissolved in Mel (2 mL) and the resulting solution was heated in a sealed vial at 60° C. for 72 h. The resulting precipitate was collected by filtration, washed with diethylether (3×5 ml) and acetone (2 ml) and dried under vacuum to afford the title compound as a yellow solid (273 mg, 0.87 mmol, 75%). These compounds react readily with any nucleophilic solvent and should be handled accordingly.

(49) The following compounds exemplify reactive matrices that may be prepared by the general methods described above:

(50) ##STR00025##

2-Fluoro-1-methyl-4-phenylpyridin-1-ium iodide (2)

(51) Prepared following the general procedures A and C, starting from 2-fluoro-4-bromopyridine. .sup.1H NMR (400 MHz, CDCl.sub.3/CD.sub.6CO): δ 9.25-9.11 (m, 1H), 8.42-8.33 (m, 2H), 8.11-8.00 (m, 2H), 7.72-7.57 (m, 3H), 4.48 (d, J=3.7 Hz, 3H).

(52) .sup.13C NMR (100 MHz, CDCl.sub.3/CD.sub.6CO): δ 161.7 (d, J=11.4 Hz), 144.6 (d, J=7.2 Hz), 132.8, 129.7, 128.1, 121.1 (d, J=3.0 Hz), 110.2 (d, J=21.5 Hz), 41.2 (d, J=5.2 Hz).

(53) Accurate mass (MALDI, m/z): calc'd for C.sub.12H.sub.11FN ([M.sup.+]): 188.0870, found m/z 188.0877.

(54) ##STR00026##

2-Fluoro-1-methyl-4-(naphthalen-2-yl)pyridin-1-ium iodide (3)

(55) Prepared following the general procedures A and C, starting from 2-fluoro-4-bromopyridine. .sup.1H NMR (400 MHz, CDCl.sub.3/CD.sub.6CO): δ 8.90 (dd, J=6.7, 4.9 Hz, 1H), 8.62 (d, J=2.0 Hz, 1H), 8.36 (dd, J=6.7, 2.1 Hz, 1H), 8.24 (dd, J=5.7, 2.1 Hz, 1H), 8-18-8.14 (m, 2H), 8.10-8.02 (m, 1H), 8.00 (dt, J=8.7, 2.2 Hz, 1H), 7.82-7.66 (m, 2H), 4.36 (d, J=3.5 Hz, 3H).

(56) .sup.13C NMR (100 MHz, CDCl.sub.3/CD.sub.6CO): δ 161.6 (d, J=11.7 Hz), 143.6 (d, J=6.8 Hz), 135.2, 133.0, 130.0, 129.9, 129.4, 128.7, 127.9, 127.7, 123.6, 121.4 (d, J=2.8), 111.3 (d, J=21.9 Hz), 41.5 (d, J=5.0 Hz).

(57) Accurate mass (MALDI, m/z): calc'd for C.sub.16H.sub.13FN ([M.sup.+]): 238.1027, found m/z 238.1028.

(58) ##STR00027##

5-(Anthracen-9-yl)-2-fluoro-1-methylpyridin-1-ium iodide (4)

(59) Prepared following the general procedures A and C, starting from 2-fluoro-4-bromopyridine. .sup.1H NMR (400 MHz, CDCl.sub.3/CD.sub.6CO): δ 8.77 (s, 1H), 8.64 (ddd, J=8.5, 5.6, 2.3 Hz, 1H), 8.59 (dd, J=4.2, 2.2 Hz, 1H), 8.18 (dt, J=8.2, 1.3 Hz, 2H), 8.05 (dd, J=8.7, 4.0 Hz, 1H), 7.61-7.52 (m, 6H), 4.29 (d, J=3.8 Hz, 3H).

(60) .sup.13C NMR (100 MHz, CDCl.sub.3/CD.sub.6CO): δ 154.4 (d, J=11.4 Hz), 145.8 (d, J=6.8 Hz), 136.0 (d, J=3.8 Hz), 137.6, 130.8, 130.4, 129.5, 127.9, 126.6, 126.4, 125.4, 115.5 (d, J=20.3 Hz), 42.9 (d, J=5.1 Hz).

(61) Accurate mass (MALDI, m/z): calc'd for C.sub.20H.sub.15FN ([M.sup.+]): 288.1183, found m/z 288.1179.

(62) ##STR00028##

2-Fluoro-1-methyl-4-(phenanthren-9-yl)pyridin-1-ium iodide (5)

(63) Prepared following the general procedures A and C, starting from 2-fluoro-4-bromopyridine.

(64) .sup.1H NMR (400 MHz, CDCl.sub.3/CD.sub.6CO): δ 8.85-8.79 (m, 1H), 8.78-8.69 (m, 2H), 8.05-8.02 (m, 2H), 8.00 (dd, J=5.0, 1.9 Hz, 1H), 7.96 (s, 1H), 7.88 (dd, J=8.2, 1.2 Hz, 1H), 7.84-7.77 (m, 2H), 7.74-7.66 (m, 2H), 4.30 (d, J=3.6 Hz, 3H).

(65) .sup.13C NMR (100 MHz, CDCl.sub.3/CD.sub.6CO): δ 163.4 (d, J=11.5 Hz), 143.9 (d, J=6.7 Hz), 132.3 (d, J=1.9 Hz), 131.7, 131.2, 130.9, 130.8, 130.1, 129.7, 128.6, 128.5, 128.4, 128.3, 125.8 (d, J=3.2 Hz), 125.6, 124.3, 123.5, 115.5 (d, J=20.5 Hz), 42.3 (d, J=5.0 Hz).

(66) Accurate mass (MALDI, m/z): calc'd for C.sub.20H.sub.16FN ([M.sup.+]): 288.1183 found m/z 288.1177.

(67) ##STR00029##

4-([1,1′:3′,1″-Terphenyl]-5′-yl)-2-fluoro-1-methylpyridin-1-ium iodide (6)

(68) Prepared following the general procedures A and C, starting from 2-fluoro-4-bromopyridine. .sup.1H NMR (400 MHz, CDCl.sub.3/CD.sub.6CO): δ 8.71-8.68 (m, 1H), 8.31 (dd, J=6.7, 2.0 Hz, 1H), 8.26 (dd, J=5.7, 2.0 Hz, 1H), 8.13-8.10 (m, 1H), 8.10 (d, J=1.7 Hz, 21H), 7.80-7.76 (m, 4H), 7.55-7.49 (m, 4H), 7.49-7.41 (m, 2H), 4.23 (d, J=3.6 Hz, 3H).

(69) .sup.13C NMR (100 MHz, CDCl.sub.3/CD.sub.6CO): δ 162.0 (d, J=11.5 Hz), 144.1 (d, J=7.0 Hz), 143.8, 139.9, 134.7 (d, J=2.2 Hz), 130.7, 129.6, 128.9, 127.9, 126.1, 122.2 (d, J=3.1 Hz), 111.4 (d, J=20.3 Hz), 41.9 (d, J=5.1 Hz).

(70) Accurate mass (MALDI, m/z): calc'd for C.sub.24H.sub.20FN ([M.sup.+]): 340.1496, found m/z 340.1496.

(71) ##STR00030##

2-Fluoro-1-methyl-4-(10-phenylanthracen-9-yl)pyridin-1-ium iodide (7)

(72) Prepared following the general procedures A and C, starting from 2-fluoro-4-bromopyridine. .sup.1H NMR (400 MHz, CDCl.sub.3/CD.sub.6CO): δ 8.87-8.84 (m, 1H), 7.98-7.94 (m, 2H), 7.70 (dd, J=8.7, 1.4, 0.8 Hz, 2H), 7.68-7.56 (m, 5H), 7.55-7.51 (m, 2H), 7.48-7.39 (m, 4H), 4.38 (d, J=3.7 Hz, 3H).

(73) .sup.13C NMR (100 MHz, CDCl.sub.3/CD.sub.6CO): δ 164.1 (d, J=11.5 Hz), 144.8 (d, J=6.8 Hz), 141.3, 138.3, 131.3, 130.0, 129.3, 129.2, 128.9, 128.7, 128.0 (d, J=3.5 Hz), 127.9, 127.8, 126.4, 125.2, 117.0 (d, J=20.9 Hz), 42.4 (d, J=5.1 Hz).

(74) Accurate mass (MALDI, m/z): calc'd for C.sub.26H.sub.19FN ([M.sup.+]): 364.1496, found m/z 364.1486.

(75) ##STR00031##

4-(10-Bromoanthracen-9-yl)-2-fluoro-1-methylpyridin-1-ium (8)

(76) Prepared following the general procedures A, B and C, starting from 2-fluoro-4-bromopyridine.

(77) .sup.1H NMR (400 MHz, CDCl.sub.3/CD.sub.6CO): δ 9.01 (t, J=5.6 Hz, 1H), 8.53 (dd, J=8.9, 1.0 Hz, 2H), 7.78 (dd, J=6.4, 1.7 Hz, 1H), 7.69 (dd, J=4.3, 1.7 Hz, 1H), 7.56 (ddd, J=9.0, 6.4, 1.2 Hz, 2H), 7.50 (dd, J=8.7, 1.1 Hz, 2H), 7.42 (ddd, J=8.8, 6.4, 1.1 Hz, 2H), 4.36 (d, J=3.7 Hz, 3H).

(78) .sup.13C NMR (100 MHz, CDCl.sub.3/CD.sub.6CO): δ 161.0 (d, J=11.5 Hz), 143.7 (d, J=6.5 Hz), 129.2, 128.7 (d, J=1.6 Hz), 128.6, 127.5, 127.2, 127.1, 126.5 (d, J=3.4 Hz), 125.4, 124.6, 124.5, 116.6 (d, J=16.9 Hz), 41.40 (d, J=5.0 Hz).

(79) Accurate mass (MALDI, m/z): calc'd for C.sub.20H.sub.14BrFN ([M.sup.+]): 366.0288, found m/z 366.0284.

(80) ##STR00032##

4-(Anthracen-9-yl)-2-fluoro-1-ethylpyridin-1-ium (9)

(81) Prepared following the general procedures A and C, starting from 2-fluoro-4-bromopyridine and using ethyl iodide as the alkylating agent. .sup.1H NMR (400 MHz, CDCl.sub.3/CD.sub.6CO): 9.32 (dd, J=6.4, 4.8 Hz, 1H), 8.67 (s, 1H), 8.12-8.07 (m, 2H), 7.96 (dd, J=6.4, 1.7 Hz, 1H), 7.79 (dd, J=4.8, 1.7 Hz, 1H), 7.64-7.61 (m, 2H), 7.55-7.45 (m, 4H), 4.92 (qd, J=7.4, 2.4 Hz, 2H), 1.81 (t, J=7.3 Hz, 3H).

(82) .sup.13C NMR (100 MHz, CDCl.sub.3/CD.sub.6CO): δ 163.3 (d, J=11.3 Hz), 143.5 (d, J=6.5 Hz), 130.9, 130.5, 129.0, 128.7, 128.1 (d, J=1.7 Hz), 127.9 (d, J=3.0 Hz), 127.8, 125.9, 124.4, 117.3 (d, J=19.5 Hz), 51.8 (d, J=4.3 Hz), 14.8 (d, J=1.0 Hz).

(83) Accurate mass (MALDI, m/z): calc'd for C.sub.21H.sub.17FN ([M.sup.+]): 302.1340, found m/z 302.1334.

(84) ##STR00033##

4-(Anthracen-9-yl)-2-fluoro-1-methylpyridin-1-ium iodide (10)

(85) Prepared following the general procedures A and C, starting from 2-fluoro-4-bromopyridine. .sup.1H NMR (400 MHz, CDCl.sub.3/CD.sub.6CO): δ 8.89 (t, J=5.5 Hz, 1H), 8.72 (s, 1H), 8.15-8.13 (m, 2H), 7.92-7.90 (m, 1H), 7.87-7.85 (m, 1H), 7.61-7.46 (m, 6H), 4.39 (d, J=3.7 Hz, 3H).

(86) .sup.13C NMR (100 MHz, CDCl.sub.3/CD.sub.6CO): δ 163.7 (d, J=11.4 Hz), 144.6 (d, J=6.8 Hz), 131.2, 130.7, 129.4, 129.1, 128.0, 127.7 (d, J=3.2 Hz), 126.2, 124.8, 117.6 (d, J=20.9 Hz), 42.4 (d, J=5.1 Hz).

(87) Accurate mass (MALDI, m/z): calc'd for C.sub.20H.sub.15FN ([M.sup.+]): 288.1183, found m/z 288.1184.

(88) ##STR00034##

4-(Anthracen-9-yl)-2-fluoro-1-methylpyridin-1-ium trifluoromethylsulfonate (11)

(89) Prepared following the general procedures A and C, starting from 2-fluoro-4-bromopyridine and using methyl triflate as the methylating agent.

(90) .sup.1H NMR (400 MHz, CDCl.sub.3/CD.sub.6CO): δ 8.81-8.73 (m, 1H), 8.72 (s, 1H), 8.15-8.02 (m, 2H), 7.84 (dd, J=6.4, 1.7 Hz, 1H), 7.78 (dd, J=4.6, 1.7 Hz, 1H), 7.62-7.43 (m, 6H), 4.38 (d, J=3.7 Hz, 3H).

(91) .sup.13C NMR (100 MHz, CDCl.sub.3/CD.sub.6CO): δ 163.7 (d, J=11.4 Hz), 144.6 (d, J=6.8 Hz), 131.0, 130.6, 129.1, 128.9, 128.4, (d, J=3.2 Hz), 126.1, 124.4, 117.2, 42.1 (d, J=5.1 Hz).

(92) Accurate mass (MALDI, m/z): calc'd for C.sub.20H.sub.15FN ([M.sup.+]): 288.1183, found m/z 288.1176.

Example 2: Determination of MALDI-MS Sensitivity of Derivatised Model Compounds

(93) To evaluate and compare the efficiency of the synthesized substances as reactive matrices for high sensitivity MALDI-MSI analysis, small molecules with different functional groups were spotted on the cortical area of a rat control brain tissue section. On the same tissue sections, rectangular regions of interest were selected in the striatal area of each brain. MALDI-MSI analysis were performed for all model substances as well as the selected striatal area with different synthesized reactive matrices. Signal to noise (S/N) values for all derivatized compounds were calculated using an in-house developed software (Kallback, P., Nilsson, A., Shariatgorji, M. & Andren, P. E. msIQuant—Quantitation Software for Mass Spectrometry Imaging Enabling Fast Access, Visualization, and Analysis of Large Data Sets. Anal Chem 88, 4346-4353 (2016)). The results are shown in Table 1 below.

(94) TABLE-US-00001 TABLE 1 Signal to noise values for detection of Added derivatized compounds mass to D.sub.4-DA target single/ Endogenous No. Name Mol. wt. molecule double DA PEA EPI HD 1 2-fluoro-1- 112.05570  92.04948 ND ND ND ND ND methylpyridin-1- ium P- toluenesulfonate 2 2-fluoro-1- 188.08700 168.08078 ND ND ND ND ND methyl-4- phenylpyridin-1- ium iodide 3 2-fluoro-1- 238.10265 218.09643  14/ND  2/ND  9  61/ND 442 methyl-4- (naphthalen-2- yl)pyridin-1-ium iodide 4 5-(anthracen-9- 288.11830 268.11208 254/332 14/30  17 156/213 637 yl)-2-fluoro-1- methylpyridin-1- ium iodide 5 2-fluoro-1- 288.11830 268.11208  20/204  2/26  6  29/365 348 methyl-4- (phenanthren-9- yl)pyridin-1-ium iodide 6 4-([1,1′:3′,1″- 340.14960 320.14338 152/341 25/105  17  93/3 200 Terphenyl]-5′- yl)-2-fluoro-1- methylpyridin-1- ium iodide 7 2-fluoro-1- 364.14960 344.14338  14/ND  2/ND  16.0  19/ND 178 methyl-4-(10- phenylanthracen- 9-yl)pyridin-1- ium iodide 8 4-(10- 366.02882 346.02259  28/18  6/5  27  72/35 433 bromoanthracen- 9-yl)-2-fluoro-1- methylpyridin-1- ium iodide 9 4-(Anthracen-9- 302.13395 282.12773 72 605 17/247  75/493 959 yl)-2-fluoro-1- ethylpyridin-1- ium iodide 10 4-(Anthracen-9- 288.11830 268.11208 115/522 16/342  79 149/827 802 yl)-2-fluoro-1- methylpyridin-1- ium iodide 11 4-(Anthracen-9- 288.11830 268.11208 204/117 19/18 111  70/46 377 yl)-2-fluoro-1- methylpyridin-1- ium trifluoromethyl sulfonate 12 2,5-Dihydroxy 154.027  1.007  5.4* ND  8.2 21.7*  22 benzoic acid (DHB) 13 α-Cyano-4- 189.042  1.007 ND ND ND ND  1.2 hydroxycinnamic acid (CHCA) ND = not detected (S/N ratio < 3) * = 10 times higher concentration of analyte used DA = dopamine; PEA = Phenethylamine; EPI = epinephrine; HD = hordenine

(95) It can be concluded from the S/N values that iodide salts of the ethylated and methylated 4-(Anthracen-9-yl)-2-fluoro-1-alkylpyridin-1-ium (entries 9 and 10) produced the best S/N values for the tested model compounds. The traditional non-reactive matrices CHCA and DHB (entries 13 and 12 respectively), which are the most widely used positive ion mode non-reactive/non-selective MALDI-MS assisting matrices for small molecules, were compared with the newly synthesized reactive matrices. The data presented in table 1 show a significant enhancement of sensitivity ranging from at least 10 times for the detection of phenethylamine to about 500 times for the analysis of dopamine when using the synthesized reactive matrices, as compared to the traditional non-reactive matrices.

Example 3: Synthesis of 4-(anthracen-9-yl)-1-(2-hydrazinyl-2-oxoethyl)pyridin-1-ium

(96) A general method for the synthesis of reactive matrices having a reactive hydrazine moiety bound at the N-pyridine position is shown in FIG. 3. The procedure is exemplified by the synthesis of 4-(anthracen-9-yl)-1-(2-hydrazinyl-2-oxoethyl)pyridin-1-ium as described below. Note however that Boc-hydrazine may be reacted with acyl halides or alkyl halides other than chloroacetyl chloride, thus enabling the provision of reactive matrices having a variety of alkyl or oxo-alkyl chains binding the hydrazine moiety to the nitrogen of the pyridine ring.

tert-butyl 2-(2-chloroacetyl)hydrazine-1-carboxylate

(97) To a stirred and cooled (0° C.) solution of tert-butyl hydrazinecarboxylate (1.98 g, 15 mmol, 2 equiv) in DCM (20 ml) and pyridine (0.79 g, 10 mmol, 1.5 equiv) was added chloro acetyl chloride (1.12 g, 10 mmol, 1.5 equiv) in DCM (5 ml). The reaction mixture was stirred over a period of 6 h and washed three times with diluted HCl (50 ml). The organic phase was dried over Na.sub.2SO.sub.4. The solvent was removed under reduced pressure, to yield the title compound as a clear oil (1.16 g, 55%).

4-(anthracen-9-yl)-1-(2-(2-(tert-butoxycarbonyl)hydrazinyl)-2-oxoethyl)pyridine-1-ium chloride

(98) A solution of 4-(anthracen-9-yl)pyridine (0.33 g, 1.29 mmol, 1 equiv., produced by general method A above), tert-butyl 2-(2 chloroacetyl)hydraxine-1-carboxylate (0.54 g, 2.58 mmol, 2 equiv) in chloroform (2 ml) was heated at 120° C. for 30 minutes. The reaction mixture was left to crystalize and the solid product was filtered and washed with ethyl acetate. It was purified with vapor diffusion crystallization (EtOH and diethyl ether). Yield of the yellow product (0.44 g, 81%).

4-(anthracen-9-yl)-1-(2-hydrazinyl-2-oxoethyl)pyridin-1-ium

(99) ##STR00035##

(100) To a stirred solution of 4-(anthracen-9-yl)-1-(2-(2-(tert-butoxycarbonyl)hydrazinyl)-2-oxoethyl)pyridine-1-ium chloride (50 mg, 0.12 mmol) in 1 ml 10% MeOH in DCM was added 1 ml of 4M HCl in 1,4-dioxane solution drop wise under nitrogen. The reaction mixture was stirred over night at room temperature under nitrogen. The precipitated form was filtered, washed with EtOAc and dried. Yield of yellow powder (36 mg, 94%). .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.35 (d, J=6.0 Hz, 2H), 8.90 (s, 1H), 8.42 (d, J=6.3 Hz, 2H), 8.25 (dd, J=8.3, 1.3 Hz, 2H), 7.60 (m, 2H) 7.55, 7.41 (d, 2H), 5.88 (s, 2H). .sup.13C NMR (101 MHz, DMSO-d.sub.6) δ 164.5, 157.2, 147.0, 131.0, 130.8, 130.3, 129.8, 129.4, 128.7, 127.9, 126.3, 124.9, 60.1.

Example 4: Use of 4-(anthracen-9-yl)-1-(2-hydrazinyl-2-oxoethyl)pyridin-1-ium as a Reactive Matrix for the MALDI-MS Analysis of 11-deoxycorticosterone

(101) The synthesised 4-(anthracen-9-yl)-1-(2-hydrazinyl-2-oxoethyl)pyridin-1-ium reactive matrix was added to a sample of 11-deoxycorticosterone on a metal plate, and the resulting derivative was analysed directly by MALDI-MS operating in positive ion mode. The resulting MALDI spectrum is shown in FIG. 4. Peaks corresponding to the reactive matrix bound to 11-deoxycorticosterone as a hydrazone can clearly be observed at m/z=640.6. No additional assisting MALDI matrix substance was required in order to obtain the MALDI spectrum.

(102) In control experiments without reactive matrix and using the traditional assisting matrix substance DHB, no signal for 11-deoxycorticosterone can be observed at the relevant sample concentrations.

Example 5: Synthesis and MALDI-MS Utility of Further Pyridinium Reactive Matrices

(103) The following compounds exemplify additional reactive matrices that may be prepared by the general methods A and/or C and those described below.

(104) ##STR00036##

4-(Dibenzo[b,d]thiophen-4-yl)-2-fluoro-1-methylpyridin-1-ium iodide (14)

(105) Prepared following the general procedures A and C, starting from 2-fluoro-4-bromopyridine.

(106) .sup.1H NMR (400 MHz, CD.sub.3CN/CD.sub.3Cl) δ 8.70 (dd, J=6.6, 5.0 Hz, 1H), 8.50 (dd, J=7.7, 1.3 Hz, 1H), 8.38-8.30 (m, 1H), 8.28 (dd, J=6.6, 2.0 Hz, 1H), 8.19 (dd, J=5.4, 2.0 Hz, 1H), 8.02-7.93 (m, 1H), 7.80 (dd, J=7.5, 1.4 Hz, 1H), 7.75 (dd, J=7.7, 7.6 Hz, 1H), 7.62-7.56 (m, 2H), 4.25 (d, J=3.6 Hz, 3H).

(107) .sup.13C NMR (101 MHz, CD.sub.3CN/CD.sub.3Cl) δ 161.0 (d, J=11.2 Hz), 143.7 (d, J=7.1 Hz), 137.4, 137.1, 136.7, 134.0, 128.6 (d, J=2.0 Hz), 127.8, 127.3, 125.2, 124.7, 124.5, 122.1 (d, J=3.6 Hz), 122.0, 121.6, 111.8 (d, J=21.4 Hz), 40.8 (d, J=5.8 Hz).

(108) MS (m/z) calc'd for C.sub.18H.sub.13FNS.sup.+ ([M.sup.+]): 294.1 found, m/z 294.1.

(109) ##STR00037##

2-Fluoro-1-methyl-4-(naphthalen-1-yloxy)pyridin-1-ium iodide (15)

(110) To a solution of 2,4-difluoropyridine (3.5 mmol) in DMF (4 mL) was added 1-naphthol (1.5 equiv) and K.sub.2CO.sub.3 (3 equiv). The resulting solution was stirred at room temperature for 18 hrs and subsequently extracted with EtOAc and brine. The intermediate 2-fluoro-4-(naphthalen-1-yloxy)pyridine was isolated by silica gel chromatography eluting with 5% EtOAc in n-Hexane. The title compound was prepared following the general procedure C, starting from 2-fluoro-4-(naphthalen-1-yloxy)pyridine. .sup.1H NMR (400 MHz, CD.sub.3CN/CD.sub.3Cl) δ 8.45 (ddd, J=7.2, 5.6, 1.3 Hz, 1H), 8.11 (d, J=8.9 Hz, 1H), 8.05-7.99 (m, 1H), 7.99-7.90 (m, 1H), 7.76 (d, J=2.5 Hz, 1H), 7.66-7.55 (m, 2H), 7.38-7.32 (m, 2H), 7.20 (dd, J=6.3, 2.6 Hz, 1H), 4.06 (d, J=3.5, 1.3 Hz, 3H).

(111) .sup.13C NMR (101 MHz, CD.sub.3CN/CD.sub.3Cl) δ 175.1 (d, J=12.5 Hz), 161.7 (d, J=273.1 Hz), 150.8, 146.4 (d, J=5.4 Hz), 135.1, 133.3, 132.7, 129.1, 129.0, 128.6, 128.0, 120.3, 119.3, 113.6 (d, J=2.5 Hz), 101.5 (d, J=25.4 Hz), 41.7 (d, J=5.0 Hz).

(112) MS (m/z) calc'd for C.sub.16H.sub.13FNO+([M.sup.+]): 254.1 found, m/z 254.2.

(113) ##STR00038##

2-(Anthracen-9-yl)-4-fluoro-1-methylpyridin-1-ium iodide (16)

(114) Prepared following the general procedures A and C, starting from 4-fluoro-2-bromopyridine.

(115) .sup.1H NMR (400 MHz, CD.sub.3CN) δ 9.26 (dd, J=7.1, 5.5 Hz, 1H), 8.96 (s, 1H), 8.30-8.22 (m, 2H), 8.09 (td, J=6.9, 3.0 Hz, 1H), 7.95 (dd, J=7.0, 3.0 Hz, 1H), 7.68-7.55 (m, 4H), 7.44 (dq, J=8.0, 1.0 Hz, 2H), 3.78 (d, J=1.0 Hz, 3H).

(116) .sup.13C NMR (101 MHz, CD.sub.3CN) δ 173.3 (d, J=280.8 Hz), 159.0 (d, J=14.1 Hz), 153.3 (d, J=13.3 Hz), 132.9, 131.9, 130.3, 130.2, 129.7, 127.2, 124.7, 123.5, 121.5 (d, J=22.1 Hz), 117.2 (d, J=22.3 Hz), 46.8.

(117) MS (m/z) calc'd for C.sub.20H.sub.15FN.sup.+ ([M.sup.+]): 288.1 found, m/z 288.1.

(118) ##STR00039##

4-(Anthracen-9-yl)-2-iodo-1-methylpyridin-1-ium chloride (17)

(119) Prepared following the general procedures A and C, starting from 4-fluoro-2-chloropyridine.

(120) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.46 (d, J=6.3 Hz, 1H), 8.91-8.82 (m, 2H), 8.29-8.18 (m, 3H), 7.65-7.59 (m, 2H), 7.59-7.52 (m, 4H), 4.54 (s, 3H).

(121) MS (m/z) calc'd for C.sub.20H.sub.151N.sup.+ ([M.sup.+]): 396.0 found, m/z 396.0.

(122) ##STR00040##

4-(Anthracen-9-ylmethyl)-2-fluoro-1-methylpyridin-1-ium iodide (18)

(123) Prepared following the general procedures A and C, starting from 2-fluoropyridin-4-ylboronic acid and 9-(bromomethyl)anthracene. .sup.1H NMR (400 MHz, CD.sub.3CN) 8.67 (s, 1H), 8.33 (dd, J=6.6, 4.8 Hz, 1H), 8.21-8.11 (m, 4H), 7.64-7.51 (m, 5H), 7.47-7.42 (m, 1H), 5.35 (s, 2H), 4.04 (d, J=3.6 Hz, 3H).

(124) .sup.13C NMR (101 MHz, CD.sub.3CN) δ 169.2 (d, J=11.2 Hz), 158.5 (d, J=280.5 Hz), 144.4 (d, J=6.6 Hz), 132.7, 131.3, 130.4, 129.3, 128.1, 128.0, 126.4, 124.8, 124.7 (d, J=3.2 Hz), 114.2 (d, J=20.5 Hz), 42.1 (d, J=5.4 Hz), 34.4 (d, J=1.8 Hz).

(125) MS (m/z) calc'd for C.sub.21H.sub.17FN.sup.+ ([M.sup.+]): 302.1 found, m/z 302.2.

(126) ##STR00041##

2-Iodo-1-methylquinolinium chloride (19)

(127) Prepared following the general procedures C, starting from 2-chloroquinoline. .sup.1H NMR (400 MHz DMSO-d.sub.6) δ 7.90 (d, J=9.4 Hz, 1H), 7.72 (dd, J=7.7, 1.6 Hz, 1H), 7.63 (ddd, J=8.7, 7.1, 1.6 Hz, 1H), 7.53 (d, J=8.5 Hz, 1H), 7.27 (ddd, J=7.7, 7.1, 1.1 Hz, 1H), 6.61 (d, J=9.5 Hz, 1H), 3.61 (s, 3H);

(128) .sup.13C NMR (101 MHz, DMSO-d.sub.6) 161.1, 139.7, 139.2, 130.8, 128.7, 121.9, 121.1, 120.1, 114.6, 29.0.

(129) MS (m/z) calc'd for C.sub.10H.sub.9N.sup.+ ([M.sup.+]): 270.0 found, m/z 270.0.

(130) ##STR00042##

2-Chloro-1-methylquinolin-1-ium trifluoromethanesulfonate (20)

(131) Prepared following the general procedure C, starting from 2-chloroquinoline and methyl trifluoromethanesulfonate. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 7.89 (d, J=9.5 Hz, 1H), 7.71 (dd, J=7.7, 1.6 Hz, 1H), 7.62 (ddd, J=8.6, 7.1, 1.6 Hz, 1H), 7.52 (dd, J=8.5, 0.9 Hz, 1H), 7.26 (ddd, J=7.9, 7.2, 1.1 Hz, 1H), 6.60 (d, J=9.4 Hz, 1H), 3.61 (s, 3H).

(132) .sup.13C NMR (101 MHz, DMSO-d.sub.6) 161.2, 139.8, 139.3, 130.9, 128.8, 122.0, 121.1, 120.2, 114.7, 29.1.

(133) MS (m/z) calc'd for C.sub.10H.sub.9ClN.sup.+ ([M.sup.+]): 178.0 found, m/z 178.0.

(134) ##STR00043##

9-Chloro-10-methylacridin-10-ium trifluoromethanesulfonate (21)

(135) Prepared following the general procedure C, starting from 9-chloroacridine and methyl trifluoromethanesulfonate. .sup.1H NMR (400 MHz, (CD.sub.3).sub.2CO) δ 9.01 (ddd, J=8.7, 1.5, 0.6 Hz, 2H), 8.97 (dt, J=9.3, 0.8 Hz, 2H), 8.60 (ddd, J=9.3, 6.8, 1.5 Hz, 2H), 8.23 (ddd, J=8.7, 6.8, 0.9 Hz, 2H), 5.12 (s, 3H); .sup.13C NMR (101 MHz, DMSO-d.sub.6/(CD.sub.3).sub.2CO) 177.3, 143.2, 134.4, 127.3, 122.6, 121.6, 116.5, 34.0.

(136) MS (m/z) calc'd for C.sub.14H.sub.11ClN.sup.+ ([M.sup.+]): 228.1 found, m/z 228.1.

(137) ##STR00044##

4-(Anthracen-9-ylethynyl)-2-fluoro-1-methylpyridin-1-ium iodide (22)

(138) 9-(Prop-1-yn-1-yl)anthracene (1 equiv), CuI (0.11 equiv), Pd(PPh.sub.3).sub.2Cl.sub.2 (0.1 equiv), 4-Bromo-2-fluoropyridine (2.7 equiv) were dissolved in dry TEA (15 mL) and the reaction mixture was stirred at 110° C. overnight. After cooling to room temperature, 100 mL of sat. NH.sub.4Cl solution was added and the aqueous phase was extracted three times with 100 mL diethyl ether. The combined organic phases were dried over Na.sub.2SO.sub.4 and concentrated under vacuum. The crude mixture was purified by column chromatography (silica gel, pentane+5% toluene and 2-5% EtOAc) to give the intermediate 4-(anthracen-9-ylethynyl)-2-fluoropyridine. The title compound was prepared following the general procedure C, starting from 4-(anthracen-9-ylethynyl)-2-fluoropyridine. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.01 (dd, J=6.6, 5.1 Hz, 1H), 8.97 (s, 1H), 8.76 (dd, J=5.6, 1.8 Hz, 1H), 8.72 (dd, J=8.7, 1.0 Hz, 2H), 8.48 (dd, J=6.5, 1.8 Hz, 1H), 8.27 (dd, J=8.4, 1.1 Hz, 2H), 7.82 (ddd, J=8.7, 6.6, 1.3 Hz, 2H), 7.71 (ddd, J=8.0, 6.6, 1.2 Hz, 2H), 4.21 (d, J=3.7 Hz, 3H).

(139) MS (m/z) calc'd for C.sub.22H.sub.15FN.sup.+ ([M.sup.+]): 312.1 found, m/z 312.1.

(140) ##STR00045##

3-(Anthracen-9-yl)-2-fluoro-1-methylpyridin-1-ium iodide (23)

(141) Prepared following the general procedures A and C, starting from 2-fluoro-3-bromopyridine.

(142) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.25-9.17 (m, 1H), 8.94 (s, 1H), 8.93-8.85 (m, 1H), 8.30-8.22 (m, 3H), 7.71 (d, J=8.7 Hz, 2H), 7.64 (ddd, J=8.3, 6.6, 1.6 Hz, 2H), 7.58 (ddd, J=8.3, 6.6, 1.6 Hz, 2H).

(143) MS (m/z) calc'd for C.sub.20H.sub.15FN.sup.+ ([M.sup.+]): 288.1 found, m/z 288.2.

(144) ##STR00046##

3-(Anthracen-9-yl)-5-fluoro-1-methylpyridin-1-ium iodide (24)

(145) Prepared following the general procedures A and C, starting from 5-fluoro-3-bromopyridine.

(146) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.66 (ddd, J=3.9, 2.4, 1.1 Hz, 1H), 9.22 (dd, J=1.30, 1.26 Hz, 1H), 8.95 (ddd, J=8.2, 2.5, 1.4 Hz, 1H), 8.91 (s, 1H), 8.26 (ddd, J=8.4, 1.4, 0.7 Hz, 2H), 7.70-7.60 (m, 4H), 7.56 (ddd, J=8.7, 6.6, 1.4 Hz, 2H), 4.45 (s, 3H).

(147) .sup.13C NMR (101 MHz, CDCl.sub.3/CD.sub.3CN) δ 161.1 (d, J=257.2 Hz), 144.6, 141.6 (d, J=7.6 Hz), 136.2 (d, J=17.6 Hz), 135.7 (d, J=36.8 Hz), 131.7, 130.7, 130.6, 129.5, 128.1, 126.6, 126.5, 125.7, 50.0.

(148) MS (m/z) calc'd for C.sub.20H.sub.15FN.sup.+ ([M.sup.+]): 288.1 found, m/z 288.2.

(149) ##STR00047##

2-(Anthracen-9-yl)-6-fluoro-1-methylpyridin-1-ium trifluoromethanesulfonate (25)

(150) Prepared following the general procedures A and C, starting from 2-fluoro-6-bromopyridine.

(151) .sup.1H NMR (400 MHz, CD.sub.3CN) 8.95 (s, 1H), 8.82 (dd, J=7.5, 7.0 Hz, 1H), 8.27 (d, J=7.7 Hz, 2H), 8.07 (dd, J=8.9, 4.5 Hz, 1H), 8.02-7.94 (m, 1H), 7.70-7.56 (m, 4H), 7.47 (d, J=8.1 Hz, 2H), 3.67 (d, J=3.9 Hz, 3H).

(152) .sup.13C NMR (101 MHz, CD.sub.3CN) δ 161.8 (d, J=281.4 Hz), 153.8 (d, J=8.4 Hz), 151.5 (d, J=12.8 Hz), 133.0, 132.0, 131.0, 130.3, 129.6, 129.4, 129.3, 127.3, 124.8, 115.5 (d, J=21.6 Hz), 39.0 (d, J=6.6 Hz).

(153) MS (m/z) calc'd for C.sub.20H.sub.15FN.sup.+ ([M.sup.+]): 288.1 found, m/z 288.2.

(154) ##STR00048##

(E)-4-(2-(Anthracen-9-yl)vinyl)-2-fluoro-1-methylpyridin-1-ium iodide (26)

(155) To an oven-dried, glass vessel was added Pd.sub.2(dba).sub.3 (0.01 eq), Cy.sub.3P (0.04 eq) and 4-(anthracen-9-ylethynyl)-2-fluoropyridine (1 eq). The vessel was sealed and flushed with N.sub.2 three times before 0.2 mL of 1,4-dioxane was added. After stirring the mixture at room temperature for 15 min, 25% aqueous formic acid (3 equiv) was added and the reaction was heated to 80° C. After 2 hours, the solvent was removed under vacuo and the residue was purified by column chromatography (pentane, 5% toluene and 2-5% EtOAc) to afford the intermediate (E)-4-(2-(anthracen-9-yl)vinyl)-2-fluoro-pyridine. The title compound was prepared following the general procedures A and C, starting from (E)-4-(2-(anthracen-9-yl)vinyl)-2-fluoro-pyridine. .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.03 (d, J=16.4 Hz, 1H), 8.91 (dd, J=6.6, 5.1 Hz, 1H), 8.74 (s, 1H), 8.56 (dd, J=6.2, 1.8 Hz, 1H), 8.41-8.35 (m, 3H), 8.23-8.15 (m, 2H), 7.67-7.56 (m, 4H), 7.36 (d, J=16.5 Hz, 1H), 4.19 (d, J=3.6 Hz, 3H).

(156) MS (m/z) calc'd for C.sub.22H.sub.17FN.sup.+ ([M.sup.+]): 314.1 found, m/z 314.2.

(157) ##STR00049##

2-Fluoro-1-methyl-4-(pyren-1-yl)pyridin-1-ium iodide (27)

(158) Prepared following the general procedures A and C, starting from 4-fluoro-2-bromopyridine.

(159) .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ 9.09 (dd, J=6.5, 5.0 Hz, 1H), 8.58 (dd, J=5.5, 1.9 Hz, 1H), 8.52 (d, J=8.1 Hz, 1H), 8.48-8.42 (m, 2H), 8.41-8.30 (m, 4H), 8.23-8.17 (m, 3H), 4.32 (d, J=3.7 Hz, 3H).

(160) MS (m/z) calc'd for C.sub.22H.sub.15FN.sup.+ ([M.sup.+]): 312.1 found, m/z 312.2.

(161) ##STR00050##

4-(Anthracen-9-yl)-2-hydrazineyl-1-methylpyridin-1-ium (28)

(162) To a solution of 4-(anthracen-9-yl)-2-fluoro-1-methyl-pyridinium iodide (10 mg, 0.036 mmol) in acetonitrile was added tert-butyl carbazate (5 mg, 0.036 mmol). The resulting mixture was stirred for 1 hour and then washed with water and ethylacetate. The combined organic layers were then evaporated with reduced vacuum to yield the crude protected intermediate. This residue was taken up in 0.2 mL of 4M HCl in dioxane and stirred at room temperature for 48 hours. The mixture was then extracted with Ethylacetate and water. The combined water layers were evaporated with reduced vacuum to yield a yellow solid (3 mg, 40%). .sup.1H NMR (400 MHz, DMSO-de) 8.69 (s, 1H), 8.23-8.06 (m, 2H), 7.91 (d, J=6.8 Hz, 1H), 7.74 (dq, J=8.4, 1.0 Hz, 2H), 7.63-7.39 (m, 4H), 6.41 (dd, J=1.9, 0.6 Hz, 1H), 6.26 (dd, J=6.8, 1.9 Hz, 1H), 3.59 (s, 3H), 3.56 (s, 4H).

(163) .sup.13C NMR (101 MHz, DMSO-de) 162.1, 150.7, 140.4, 133.6, 131.2, 129.1, 128.6, 127.7, 126.8, 125.9, 121.1, 108.9, 37.28.

(164) MS (m/z) calc'd for C.sub.20H.sub.18N.sub.3.sup.+ ([M].sup.+) m/z 300.2, found m/z 300.2

(165) To evaluate the efficiency of the synthesized substances as reactive matrices for high sensitivity MALDI-MSI analysis, small molecules with primary amine, and/or phenolic groups were used as test substances. Two concentrations (0.1 mg/ml and 0.01 mg/ml) of serotonin (5-HT), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), 3-O-Methyldopa (3-OMD) and γ-aminobutyric acid (GABA) were deposited (0.5 μl) on a MALDI metal target. The reactive matrix solutions were prepared by dissolving the synthesized compounds in 70% acetonitrile (4.4 mM). An automated pneumatic sprayer (TM-Sprayer, HTX technologies) was used to spray 5 ml of heated reagent over the spotted standards. The nozzle temperature was set at 80° C. and the reagent was sprayed in 30 passes at a flow of 80 μl/min. MSI data was acquired in positive ionization mode on a MALDI-FT-ICR (Solarix XR 7T-2Ω, Bruker Daltonics) instrument by rastering across the spots at a spatial resolution of 250 μm. Signal to noise (S/N) values for all derivatized compounds were extracted from the average spectrum of each spot using an in-house developed software (msIQuant). The results are shown in Table 2 below.

(166) TABLE-US-00002 TABLE 2 Analyte of Interest Test reactive matrix name 5HT 3-OMD DA DOPAC GABA 4-(Dibenzo[b,d]thiophen-4-yl)-2-fluoro-1-methylpyridin-1- Y Y Y Y Y ium iodide (14) 2-Fluoro-1-methyl-4-(naphthalen-1-yloxy)pyridin-1-ium Y Y Y Y Y iodide (15) 2-(Anthracen-9-yl)-4-fluoro-1-methylpyridin-1-ium iodide (16) Y Y Y Y Y 4-(Anthracen-9-yl)-2-iodo-1-methylpyridin-1-ium iodide (17) N Y Y Y Y 4-(Anthracen-9-ylmethyl)-2-fluoro-1-methylpyridin-1-ium Y Y Y Y Y iodide (18) 2-Iodo-1-methylquinolin-1-ium chloride (19) Y Y Y Y Y 2-Chloro-1-methylquinolin-1-ium trifluoromethanesulfonate Y Y Y Y N (20) 9-Chloro-10-methylacridin-10-ium trifluoromethanesulfonate Y Y Y Y N (21) 4-(Anthracen-9-ylethynyl)-2-fluoro-1-methylpyridin-1-ium Y Y Y Y Y iodide (22) 3-(Anthracen-9-yl)-2-fluoro-1-methylpyridin-1-ium iodide (23) Y Y Y Y Y 3-(Anthracen-9-yl)-5-fluoro-1-methylpyridin-1-ium iodide (24) Y Y Y Y Y 2-(Anthracen-9-yl)-6-fluoro-1-methylpyridin-1-ium Y Y Y Y Y trifluoromethanesulfonate (25) (E)-4-(2-(Anthracen-9-yl)vinyl)-2-fluoro-1-methylpyridin-1- Y Y Y Y Y ium iodide (26) 2-Fluoro-1-methyl-4-(pyren-1-yl)pyridin-1-ium iodide (27) Y Y Y Y Y Y = S/N greater than 3; N = S/N less than 3.

(167) As can be seen from Table 2 all synthesized reactive matrices were effective in promoting the ionization and desorption of a range of analytes.

Example 6: Use of Bromo-Pyrylium Reactive Matrices for the MALDI-MSI Detection of Drugs in Brain Tissue Sections

(168) Charge-tagging through pyrylium derivatization has been proven to increase the sensitivity for MALDI and DESI-MSI analysis of primary amine containing small molecules. Since such derivatization adds just carbon and hydrogen to the target compounds, the resulted isotopic patterns of the products are not distinctive from those species which are not derivatized but ionized, desorbed and detected unselectively. By addition of bromine to the substituted pyrylium ion, this results in a distinctive isotopic pattern and mass defect for derivatized substances which makes them distinguishable from underivatized compounds.

(169) Material and Reagents

(170) Tetrafluoroborate salts of 2,4,6-triphenylpyrylium (TPP) and 2-(4-bromo-phenyl)-4,6-diphenyl-pyranylium (Br-TPP), triethylamine (TEA), and all other chemicals were purchased from Sigma-Aldrich (Stockholm, Sweden) unless otherwise stated and were used without further purification. Water, methanol, and trifluoroacetic acid (TFA) were obtained from Merck (Hohenbrunn, Germany).

(171) Animal Experiments

(172) All animals were housed in air-conditioned rooms (with a 12 h dark/light cycle) at 20° C. and 53% humidity, with access to food and water ad libitum. Experiments were performed in accordance with the European Communities Council Directive of Nov. 24, 1986 (86/609/EEC) on the ethical use of animals and were approved by the local ethical committee at the Karolinska Institute (N350/08 and N40/13).

(173) Adult male C57BL/6 mice (3 months old, Charles River Laboratories, Köln, Germany) were used for experiments with fluvoxamine. Fluvoxamine was dissolved in 0.25% tween 80/saline and injected i.p. at a dose of 40 mg/kg. Mice were sacrificed by cervical dislocation 30 min after fluvoxamine administration. Vehicle control was obtained by injecting mouse with a volume of saline equal to that administered in the experimental treatment. All brains were immediately removed, snap frozen, and stored at −80° C. until further analysis.

(174) Tissue Preparation and Derivatization

(175) The frozen brain tissues were cut using a cryostat-microtome (Leica CM3050S; Leica Microsystems, Welzlar, Germany) at a thickness of 14 μm, thaw-mounted onto conductive indium tin oxide (ITO) glass slides (Bruker Daltonics), and stored at −80° C. Sections were dried gently under a flow of nitrogen and desiccated at room temperature for 15 min, after which they were imaged optically using a photo scanner (Epson perfection V500).

(176) Brain samples were then coated with TPP and Br-TPP. Derivatization solutions were prepared by dissolving 8 mg of TPP or Br-TPP in 6 ml of 80% MeOH containing 3.5 μL of TEA. An automated pneumatic sprayer (TM-Sprayer; HTX Technologies, Carrboro, N.C.) was used to spray warm reagent over the tissue sections. The nozzle temperature was set at 80° C., and the reagent was sprayed in 30 passes over the tissue sections at a linear velocity of 110 cm/min with a flow rate of 80 μL/min. Samples were incubated for 15 min (dried by nitrogen flow every 5 m) in a chamber saturated with vapour from a 50% methanol solution before analysis.

(177) Mass Spectrometry

(178) All MALDI-MSI experiments were performed using a MALDI-TOF/TOF (Ultraflextreme, Bruker Daltonics, Bremen, Germany) mass spectrometer with a Smartbeam II 2 kHz laser in positive ion mode. The laser power was optimized at the start of each run and then held constant during the MALDI-MSI experiment. Fleximaging v 4.0 build 32 (Bruker Daltonics, Billerica, Mass.) was used for normalization and visualization of MALDI-MSI data.

(179) Results

(180) TPP and its brominated derivative, Br-TPP, were used to map the localization of fluvoxamine in mouse brain tissue sections, see FIG. 5. FIG. 5 shows the MALDI-MSI relative abundance and spatial distribution of TPP (a, b) and Br-TPP (d, e) derivatized fluvoxamine (m/z 609.3 and 687.2) in control (a, d) and administered (b, e) brain tissue sections without any assisting matrix. FIG. 5 also shows the average spectra of TPP (c) and Br-TPP (f) treated fluvoxamine-administered tissue section. Data are shown using a rainbow scale, normalized against the total ion count. Scale bar, 5 mm; spatial resolution=100 μm.

(181) Fluvoxamine is a selective serotonin reuptake inhibitor (SSRI) that was originally developed as an antidepressant but is commonly used for treatment of anxiety disorders. The primary amine functional group of fluvoxamine reacts selectively with both TPP and Br-TPP. Brain tissue sections from control and fluvoxamine administered mice were derivatized by non-brominated TPP (a, b) and brominated Br-TPP (d, e). No interfering signal was detected in control brain tissue section using TPP (a) and Br-TPP (d) while derivatized fluvoxamine was detected at m/z 609.3 using TPP (b) and at m/z 687.2 using Br-TPP (e) without any assisting matrix. Similar distribution of fluvoxamine, mostly localized in cortex and cerebellum, was found using both derivatization agents. The isotopic pattern of TPP derivatized fluvoxamine represents a typical non-labelled compound (c) while bromine containing Br-TPP derivatized fluvoxamine demonstrates a specific isotopic pattern that can be easily distinguished from signals corresponding to non-derivatized substances.