Compositions, methods and systems for the synthesis and use of imaging agents.

DIVISIONAL SUB-DIRECTOR OF PATENT FUND EXAMINATION BIOTECHNOLOGICAL, PHARMACEUTICAL AND CHEMICAL AREAS

EMELIA HERNÁNDEZ PRIEGO

<img file="MX367382B_D0001.tif" />

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EMELIA HERNANDEZ PRIEGO | 00001000000405397295 | Administration Service

Tr¡butar¡a | 56 || MX / 2019/70769 | MX / a / 2016/010906 | Normal patent title with divisional PCT | 1220 | RRGO | Page (s)

2 | kp8piWE9z6X + kGxEcpA1 ITalpOA =

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MX 201970769

COMPOSITIONS, METHODS AND SYSTEMS FOR SYNTHESIS AND USE

OF TRAINING AGENTS

Field of the Invention

The present invention relates to systems, compositions, methods and apparatus for synthesizing tracing agents and precursors thereof.

Background of the Invention

Heart failure (HF) is defined as the inability of the heart to supply enough blood flow to the peripheral organs. It can be characterized by a hyperadrenergic state in which elevated systemic levels of norepinephrine (NE) and an increase in catecholamine infiltration are detected. Each year the number of people affected by this pathology increases, and it is a common terminal stage of many heart diseases and conditions including myocardial infarction, pressure / volume overload, viral myocarditis, toxic cardiomyopathy, valve failure and others anomalies The resulting myocardial lesion, together with the activation of cytokines and neurohormones, stimulates the remodeling of the chamber, which is the initial phase of the development of HF. The remodeling process produces a reduction in global myocardial efficacy and the consequent progress to

Clinical IC. To date, there is no cure for the condition, therefore, early diagnosis is a decisive factor in its treatment and long-term prognosis. Therefore, a tracing agent that identifies subjects with early HF would allow the treatment to be applied and improve the lifestyle in patients suffering from the condition.

Therefore, better methods, systems and apparatus are needed to synthesize and administer tracing agents (eg, for heart tomography). In addition, although there are numerous synthetic methods for preparing trace agents for use in PET, these usually require several synthetic steps (e.g., marking a compound with a tracer moiety) and / or purification, usually have low chemical reproducibility and / or low chemical efficiency. Therefore, better synthetic methods and compositions are needed to prepare these compounds.

Brief Description of the Invention

The invention provides, in a broad sense, methods for synthesizing tracer agents and their precursors, compounds (including salt forms) that are precursors of tracer agents or tracer agents, and methods for using them.

In one aspect, the invention provides compositions.

In some embodiments, a composition comprises a compound comprising the formula (II):

<img file="MX367382B_D0002.tif" />

(ID, or a salt, free base or combination thereof, where R¹ it is alkyl, haloalkyl, alkynyl, alkenyl, heteroalkyl, cycloalkyl, aryl, heteroaryl, arylalkyl, heterocyclyl or heteroarylalkyl, each optionally substituted; every R² it can be identical or different and is hydrogen or a nitrogen protecting group; R³, R⁴, R^s and R⁶ they can be identical or different and are individually hydrogen, Ci-C6 alkyl / heteroalkyl, halide, -OR⁷, -MR⁷, -N (R⁷) 2 or -C (= O) R⁸, each optionally substituted; every R⁷ it may be identical or different and is hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, haloalkyl, aryl or heteroaryl, each optionally substituted; every R⁸ it can be identical or different and is hydrogen, alkyl, heteroalkyl, cycloalkyl, haloalkyl, heterocyclyl, aryl, heteroaryl, -OH, alkoxy, -NH₂, alkylamino, -SH or alkylthiol, each optionally substituted; m is an integer between 1 and 12, inclusive; and n is an integer between 1 and 4, inclusive.

In some embodiments, a compound of formula (II) comprises the structure of formula (IV):

<img file="MX367382B_D0003.tif" />

(IV), or a salt, free base or combination thereof.

In some embodiments, a compound of formula (IV) comprises formula (III):

<img file="MX367382B_D0004.tif" />

where Χθ is a contraanion. In some embodiments, X® is halide, phosphate, sulfate, trifluoroacetate, tolunosulfonate, acetate, formate, citrate, ascorbate, mesylate (methanesulfonate) or benzoate.

In some embodiments, a compound of formula (II) comprises the formula:

NR²

<img file="MX367382B_D0005.tif" />

N (R²)₂

In some embodiments, for any of the compositions described above, at least one R² It is not hydrogen.

In some embodiments, a compound of formula (II) comprises the formula:

<img file="MX367382B_D0006.tif" />

R⁴ NR²

<img file="MX367382B_D0007.tif" />

R⁶ or a salt, free base or combination thereof.

In some embodiments, a compound of formula (II) comprises the formula:

NH

<img file="MX367382B_D0008.tif" />

or a salt, free base or combination thereof.

In some modalities, m is 3. In some modalities, n is 1. In some modalities, R³ is Br. In some modalities, R¹ it is Cj-Ce alkyl, haloalkyl or aryl. In some modalities, R¹ it is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl or hexyl. In some modalities, R¹ It is haloalkyl. In some modalities, R¹ It is CF3. In some modalities, R¹ it is phenyl (Ph), optionally substituted. In some modalities, R¹ is 4-CH3Ph, 2,4,6- (CH₃) ₃CsH2 or CgíLX, where X is halide. In some modalities, m is an integer between 1 and 10, inclusive; or between 1 and 8, inclusive; or between 1 and 6, inclusive. In some modalities, R⁴, R⁵ and R⁶ they are hydrogen; and R³ it is halide (e.g., Br). In some embodiments, the composition comprises a salt of the compound of formula (II). In some embodiments, the salt is a pharmaceutically acceptable salt. In some modalities, at least one R² It is tbutyloxycarbonyl.

In one aspect, the invention provides a compound comprising the formula:

Brx, CN ⁰ or a salt thereof, where m is an integer between 2 and 12, inclusive. In certain modalities, m is an integer between 3 and 12, inclusive. In a₅ mode, m is 3.

In one embodiment, the invention provides a compound having the structure:

BK / Gk / N or

In one aspect, the invention provides a compound comprising the formula:

^Br\ / Hx ^ NH₂⁵ or a salt, free base or combination thereof, where m is an integer between 2 and 12, inclusive. In certain modalities, m is an integer between 3 and 12, inclusive. In one mode, tn is 3.

In one embodiment, the invention provides a compound having the structure or a free base, salt or combination thereof.

In one aspect, the invention provides a compound comprising the formula:

ΒΓΝ '

R 'or a salt, free base or combination thereof; where every R²it can be identical or different and is hydrogen or a nitrogen protecting group; and m is an integer between 2 and 12, inclusive. In certain modalities, m is an integer between 3 and 12, inclusive. In one mode, tn is 3.

In certain embodiments, the invention provides a compound comprising the formula:

Br ·

0'

NH

In certain embodiments, the invention provides a compound that has the structure

NR²

HO

<img file="MX367382B_D0009.tif" />

N NHR²

R² where R^z It can be identical or different and is hydrogen or a nitrogen protecting group.

In one embodiment, the invention provides a compound having the structure.

NBoc

<img file="MX367382B_D0010.tif" />

N NHBoc

H

In one embodiment, the invention provides a compound having the structure.

NBoc

<img file="MX367382B_D0011.tif" />

NH₂

Boc

In one aspect, the invention provides a method comprising reducing a compound comprising the formula:

<img file="MX367382B_D0012.tif" />

or a salt thereof, where m is an integer between 3 and 12, inclusive, with a reducer in suitable conditions to form a compound comprising:

νη₂ or a salt, free base or combination thereof. In one mode, m is 3. In one mode, the reducer is BH₃.

In one aspect, the invention provides a method comprising reacting a compound comprising the formula:

or a salt, free base or combination thereof, where m is an integer between 2 and 12, inclusive; under suitable conditions to form a compound comprising the formula:

Brn

R² '0' or a salt, free base or combination thereof, where each R²it can be identical or different and is hydrogen or a nitrogen protecting group; and m is an integer between 2 and 12, inclusive. In certain modalities, m is an integer between 3 and 12, inclusive. In one embodiment, m is 3. In one embodiment, the reaction step comprises reacting a compound comprising the formula:

<img file="MX367382B_D0013.tif" />

with a compound of formula:

<img file="MX367382B_D0014.tif" />

In one embodiment, the compound comprising the

<img file="MX367382B_D0015.tif" />

In one embodiment, the compound comprising the formula:

<img file="MX367382B_D0016.tif" />

It is of formula:

<img file="MX367382B_D0017.tif" />

In other aspects, the invention provides compositions comprising one or more of any of the

NR²

TO ₂

NN (R²)2

R² previous compounds, including their free bases, their salts and combinations.

In another aspect, the present invention provides methods for forming compounds. In a first embodiment, a method comprises reacting a compound comprising the formula (II):

R⁴

OO ¡i ηΓ n 's^z x JL .T

R¹'Ό ^ Ο^/γ^χΕ⁵R⁶ or a salt, free base or combination thereof, under suitable conditions to form a compound comprising the formula (iv) =

R⁴ NH

<img file="MX367382B_D0018.tif" />

R⁶ (IV), or a salt, free base or combination thereof, where R¹ it is alkyl, heteroalkyl, cycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, alkenyl, alkynyl, heterocyclyl or haloalkyl, each optionally substituted, · each R² it can be identical or different and is hydrogen or a nitrogen protecting group, provided that at least one R² is not hydrogen; R³, R⁴, R^s and R^s they can be identical or different and are individually hydrogen, Ci-C6 alkyl, heteroalkyl, halide, -OR⁷, -MR⁷, -N (R⁷) 2 or 12

C (= O) R⁸, each optionally substituted; every R⁷ it may be identical or different and is hydrogen, alkyl, heteroalkyl, cycloalkyl, haloalkyl, aryl, heteroaryl or heterocyclyl, each optionally substituted; every R⁸it can be identical or different and is hydrogen, alkyl, heteroalkyl, cycloalkyl, haloalkyl, heterocyclyl, aryl, heteroaryl, -OH, alkoxy, -NH₂, alkylamino, -SH or alkylthiol, each optionally substituted; m is an integer between 1 and 12, inclusive; and n is an integer between 1 and 4, inclusive.

In another embodiment, a method comprises reacting a compound comprising the formula (II);

R⁴ NR² ° w ° ^RVSr ^^^{N (R2}>²^r2^Re (II) or a salt, free base or combination thereof, under suitable conditions to form a compound comprising the formula (I) =

R⁴ NR²

R3 1 n Jl '^^XM ^ N ^ N (R²)₂í-1 Jl ^ JL »2 f'Vvr⁵^R6 (I), or a salt, free base or combination thereof, where R¹ it is alkyl, heteroalkyl, cycloalkyl, aryl, heteroaryl, arylalkyl, heterocyclyl, heteroarylalkyl, alkenyl, alkynyl or haloalkyl, each optionally substituted; every R² it can be identical or different and is hydrogen or a nitrogen protecting group; R³, R⁴, R⁵ and R⁶they can be identical or different and are individually hydrogen, Ci-Cg alkyl, heteroalkyl, halide, -0R⁷, -MR⁷, -N (R⁷) 2 or -C (= O) R⁸, each optionally substituted; every R⁷ it may be identical or different and is hydrogen, alkyl, heteroalkyl, cycloalkyl, haloalkyl, aryl, heteroaryl or heterocyclyl, each optionally substituted; every R⁸ it can be identical or different and is hydrogen, alkyl, heteroalkyl, cycloalkyl, haloalkyl, heterocyclyl, aryl, heteroaryl, -OH, alkoxy, -NH2, alkylamino, -SH or alkylthiol, each optionally substituted; m is an integer between 1 and 12, inclusive; and n is an integer between 1 and 4, inclusive.

In some embodiments, the method further comprises reacting the compound comprising the formula (I):

R⁴

NR²^ N ^ N (R²)₂

N

R² or a salt, free base or combination thereof, provided that at least one R² other than H, under suitable conditions to form a compound comprising the formula (V):

or a salt

<img file="MX367382B_D0019.tif" />

Free base or combination of this.

In yet another embodiment, a method comprises reacting a compound comprising the formula (IV):

<img file="MX367382B_D0020.tif" />

(IV) or a salt, free base or combination thereof under suitable conditions to form a compound comprising the formula

R⁴ NH νηρΤ'Ν NH_{two F}^₀TO₁Ar₅ H ^r6 (V), or a salt, free base or combination thereof, where R¹ it is alkyl, heteroalkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, arylalkyl, heteroarylalkyl, alkenyl, alkynyl or haloalkyl, each optionally substituted; R³, R⁴, R^s and R⁶ they can be identical or different and are individually hydrogen, CVCg alkyl, heteroalkyl, halide, -0R, -SR, -N (R⁷)₂ or -C (= O) R⁸, each optionally substituted; every R⁷ it may be identical or different and is hydrogen, alkyl, heteroalkyl, cycloalkyl, haloalkyl, aryl, heteroaryl or heterocyclyl, each optionally substituted; every R⁸ it can be identical or different and is hydrogen, alkyl, heteroalkyl, cycloalkyl, haloalkyl, heterocyclyl, aryl, heteroaryl, -OH, alkoxy, -NH_2/alkylamino, -SH or alkylthiol, each optionally substituted; m is an integer between 1 and 12, inclusive; and n is an integer between 1 and 4, inclusive.

In some embodiments, a compound of formula (II) comprises formula (IV):

<img file="MX367382B_D0021.tif" />

or a salt, free base or combination thereof.

In some embodiments, a compound of formula (IV) comprises formula (III):

<img file="MX367382B_D0022.tif" />

where Χθ is a contraanion. In some embodiments, X® is halide, phosphate, sulfate, trifluoroacetate, tolunosulfonate, acetate, formate, citrate, ascorbate, mesylate (methanesulfonate) or benzoate.

In some embodiments, a compound of formula (II) comprises the formula:

NR²

<img file="MX367382B_D0023.tif" />

East.

or a salt, free base or combination of

In some modalities, at least one R² it is not hydrogen, optionally, where at least one R² It is t-butyloxycarbonyl. In some embodiments, the compound of formula (II) comprises the formula:

R⁴

NR²

<img file="MX367382B_D0024.tif" />

NHR²

R⁴

NR²

<img file="MX367382B_D0025.tif" />

NR²

R⁴

R1

o., or 'V'

R¹' '0-^0

<img file="MX367382B_D0026.tif" />

NH₂

R² n

R⁵R⁶ a salt, free base or combination of this.

In some embodiments, a compound of formula (II) comprises the formula:

NH

<img file="MX367382B_D0027.tif" />

or a salt, free base or combination thereof.

In some modalities, m is 3. In some modalities, m is an integer between 3 and 12, inclusive. In some modalities, R³ it's halide; and R⁴-R⁶ They are hydrogen. In some modalities, R³ is Br. In some modalities, R¹ it is C1-C6 alkyl, haloalkyl or aryl. In some modalities, R¹it is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl or hexyl. In some modalities, R¹ It is haloalkyl. In some modalities, R¹ It is CF3. In some modalities, R¹ it is phenyl (Ph), optionally substituted. In some modalities, R¹ it's 4-CH₃C_fiH₄, 2,4,6- (CH₃) ₃C₆H₂ or C₆H₄X, where X is halide. In some modalities, n is 1. In some modalities, m is an integer between 1 and 10, inclusive; or between 1 and 8, inclusive; or between 1 and 6, inclusive. In some modalities, F is isotopically enriched with¹⁸F.

In one embodiment, a compound of formula (II) comprises the formula:

NBoc Br

II .1 h

Br

In one aspect, a compound of formula (II) comprises the formula:

<img file="MX367382B_D0028.tif" />

NBoc

<img file="MX367382B_D0029.tif" />

In one aspect, a compound of formula (II) comprises the formula

NBoc

<img file="MX367382B_D0030.tif" />

N NHBoc

H

In one aspect, a compound of formula (II) comprises the formula:

NBoc

<img file="MX367382B_D0031.tif" />

In some embodiments, a compound of formula (II) comprises the formula:

<img file="MX367382B_D0032.tif" />

In some embodiments, the compound comprising formula (I), formula (II), and / or formula (IV) is provided as a solution in a solvent.

In some embodiments, suitable conditions for deprotection comprise exposing the compound of formula (I) and / or formula (II) to an acid or an acidic environment. In some embodiments, the acid is hydrochloric acid, formic acid, sulfuric acid, benzoic acid, acetic acid, trifluoroacetic acid, p-toluenesulfonic acid, phosphoric acid or methanesulfonic acid. An acidic environment may be, for example, a pH less than or equal to 4, less than or equal to 3, less than or equal to 2, or less than or equal to 1.

In some embodiments, suitable conditions comprise that the reaction takes place at a temperature greater than or equal to room temperature. In some embodiments, suitable conditions for deprotection and / or fluorination may comprise a temperature between about 100 ° C and about 150 ° C, including a temperature of about 100 ° C.

In some embodiments, suitable conditions comprise that the reaction takes place at a temperature of

<td>approximately</td><td> 50</td><td>° C,</td><td>OR</td><td>approximately</td><td> 60</td><td>'C,</td><td>OR</td>

<td>approximately</td><td> 70</td><td>° c,</td><td>or</td><td>approximately</td><td> 80</td><td>'C,</td><td>or</td>

<td>approximately</td><td> 90</td><td>° C,</td><td>or</td><td>approximately</td><td> 100</td><td>° c.</td><td>or</td>

<td>approximately</td><td> 110</td><td>° C,</td><td>or</td><td>approximately</td><td> 120</td><td>'C,</td><td>or</td>

<td>approximately</td><td> 150</td><td>° c,</td><td>or</td><td>approximately</td><td> 170</td><td>'C,</td><td>or</td>

<td>approximately</td><td> 200</td><td>'C,</td><td>or</td><td>approximately</td><td> 225</td><td>'C,</td><td>or</td>

approximately 250 ° C for a period approximately less than or equal to 5 minutes, or approximately less than or equal to 10 minutes, or approximately less than or equal to 20 minutes, or approximately less than or equal to 30 minutes.

In some embodiments, suitable conditions comprise a pH of the solution less than or equal to about 13, or less than or equal to about 12, or less than or equal to about 11. In some embodiments, suitable conditions comprise a pH of the solution comprised. between about 8 and about 9, or between about 8 and about 10, or between about 7 and about 8. In some embodiments, conditions suitable for fluorination comprise a pH in the range of about 8-13, about 9-13, about 10-13 or about 10-12.

In some embodiments, the solvent is benzene, toluene, xylene, diethyl ether, glycol, diethyl ether, hexane, pentane, methylene chloride, chloroform, dioxane, tetrahydrofuran, ethyl acetate, water or mixtures thereof. In some embodiments, the compound comprising formula (V) is isolated using column chromatography.

In some embodiments, the reaction step comprises exposing a compound comprising formula (IV) to a source of fluoride. In some embodiments, the fluoride source is isotopically enriched with^ieF. In some embodiments, the source of fluoride is NaF or KF.

In some embodiments, suitable conditions further comprise exposing a compound comprising formula (II) or formula (IV) to a source of fluoride in the presence of an ammonium salt or a bicarbonate salt. In some embodiments, the molar ratio of ammonium salt or bicarbonate salt to the compound of formula (IV) is less than or equal to about 10: 1, or less than or equal to about 9: 1, or less than or equal to about 8 : 1, or less than or equal to about 7: 1, or less than or equal to about 6: 1, or less than or equal to about 5: 1, or less than or equal to about 4: 1, or less than or equal to about 3 : 1, or less than or equal to about 2: 1, or less than or equal to about 1: 1. In some embodiments, the ammonium salt is an ammonium bicarbonate type salt, an ammonium hydroxide type salt, an ammonium acetate type salt, an ammonium lactate type salt, an ammonium trifluoroacetate type salt, a ammonium methanesulfonate type salt, a p-toluenesulfonate type ammonium salt, an ammonium nitrate type salt, an ammonium iodide type salt or an ammonium bisulfate type salt. In some embodiments, the bicarbonate salt is a tetraalkylammonium bicarbonate. In some embodiments, the ammonium salt or bicarbonate salt comprises the formula: R₄NHCO₃where R₄ It is alkyl. In some embodiments, the reaction is carried out in the presence of a crypto.

In embodiments, a method comprises reacting a compound comprising the formula (XI):

R⁴

NR²

N (R²)₂

JL R²

R^and (XI) or a salt, free base or combination thereof, under suitable conditions to form a compound comprising the formula (II):

R⁵ (II), or a salt, free base or combination thereof, where R¹ it is alkyl, heteroalkyl, cycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, alkenyl, alkynyl, heterocyclyl or haloalkyl, each optionally substituted; every R² it can be identical or different and is hydrogen or a nitrogen protecting group, provided that at least one R² is not hydrogen; R³, R⁴, R^s and R⁶ they can be identical or different and are individually hydrogen, Ci-C alkyl_and, heteroalkyl, halide, -OR⁷, -MR⁷,

-N (R⁷)₂ or -C (= O) R⁸, each optionally substituted; every R⁷it may be identical or different and is hydrogen, alkyl, heteroalkyl, cycloalkyl, haloalkyl, aryl, heteroaryl or heterocyclyl, each optionally substituted; every R⁸it can be identical or different and is hydrogen, alkyl, heteroalkyl, cycloalkyl, haloalkyl, heterocyclyl, aryl, heteroaryl, -OH, alkoxy, -NH₂, alkylamino, -SH or alkylthiol, each optionally substituted; m is an integer between 1 and 12, inclusive; and n is an integer between 1 and 4, inclusive.

In yet another aspect, the invention provides particular salts of tracing agents and / or their precursors. In one embodiment, a salt comprises the formula (VI):

NH₂® θΧ (VI) where X® is formate.

In another embodiment, a salt comprises the formula (VII): NH₂® θΧ ^{ΒΓνΛ nh}2

II JH (vile) where X® is ascorbate.

In some embodiments, the salt is a citrate salt or a trifluoroacetate salt comprising the cation of formula (VI) or (VII).

In some embodiments, the fluoride of a salt is isotopically enriched with ¹⁸F.

In some embodiments, a pharmaceutically acceptable composition is provided comprising a salt such as those described herein and optionally a pharmaceutically acceptable excipient.

In some embodiments, a kit is provided comprising a salt or a composition such as those described herein and instructions for use.

In another aspect, methods for performing a tomography are provided. In one embodiment, a method for performing a tomography of a subject comprises administering a dose of a pharmaceutically acceptable composition, comprising a tracing agent, including salts thereof, such as those described herein, where fluorine is isotopically enriched with¹⁸F, and optionally a pharmaceutically acceptable excipient, to a subject; and acquire at least one image of a portion of the subject. In some embodiments, the maximum dose of the tracing agent is approximately less than or equal to 15 mCi, less than or equal to 14 mCi, less than or equal to 13 mCi, less than or equal to 12 mCi, less than or equal to 11 mCi, or less than or equal to 10 mCi.

In one aspect, the invention provides the use of a salt such as those described herein to perform a tomography of a portion of a subject.

In some embodiments, a method is provided for performing a tomography of a subject comprising administering a dose of a compound comprising the formula:

NH η N NH? AJ^h or a free base, pharmaceutically acceptable salt or combination thereof, to a subject, where the maximum dose of the compound administered to the subject is approximately less than or equal to 15 mCi; and acquire at least one image of a portion of the subject.

In some embodiments, a method is provided for detecting the norepinephrine transporter (NET) in a portion of a subject, the method comprising administering a dose of a compound comprising the formula:

<img file="MX367382B_D0033.tif" />

or a free base, pharmaceutically acceptable salt or combination thereof, to a subject, where the maximum dose of the compound administered to the subject is less than about 14 mCi; and acquire at least one image of the subject's portion, where the image detects the NET in the subject.

In some embodiments, the maximum dose of the compound administered to the subject is approximately less than or equal to 13 mCi, is between about 10 mCi and about 13 mCi, or is between about mCi and about 10 mCi.

In some modalities, the acquisition step uses positron emission nomography. In some form, the portion of the subject from which images are being acquired is at least a portion of the cardiovascular system, the heart or is at least a portion of the heart.

In some embodiments, the method further comprises determining the presence or absence of a cardiovascular disease or condition in the subject.

In some embodiments, the compound is provided to be administered in a solution comprising between about 1% and about 10% ethanol and between about 25 mg / mL and about 75 mg / mL of ascorbic acid.

In some embodiments, the method further comprises administering a second dose of the compound to the subject at a time after the first dose; and acquire at least one image of the portion of the subject after administering the second dose of the compound. In some embodiments, the method further comprises comparing the image or images acquired after the first dose with the image or images acquired after the second dose; and determine the presence or absence of differences between cardiac sympathetic innervation at the time of administration of the first and second doses of the compound to the subject.

In some modalities, the presence of NET indicates the presence of a condition. In some modalities, the condition is a tumor.

In some embodiments, the detection comprises determining the level, density, location and / or function of NET in the portion of the subject.

In some embodiments, the method further comprises assessing the sympathetic cardiac innervation in the subject.

In some embodiments, the determination step comprises determining the level, density, location or function of NET in the subject portion.

In some modalities, dynamic image tomographic data is used to differentiate changes in local or global blood flow from changes in the global or local NET function or distribution.

In some embodiments, the method further comprises providing tomographic data using another tracing agent and determining blood flow based on tomographic data to differentiate local or global blood flow from local or global changes in NET function or distribution.

<td>In</td><td>some modalities,</td><td>the method</td><td>also includes</td>

<td>evaluate</td><td>sympathetic innervation</td><td>cardiac in</td><td>the subject.</td>

<td>In</td><td>Some modalities</td><td>at least</td><td>a portion of</td>

Compound is present as a pharmaceutically acceptable salt. In some embodiments, the salt is a formate salt or the ascorbate salt of the compound. In some embodiments, the salt is the citrate salt or the trifluoroacetate salt of the compound.

Brief Description of the Figures

Figure 1 shows an example of a fluorination reaction- [^{1 B}F] nucleophilic which employs a precursor of the tracer agent and a source of fluoride to form a tracer agent of the invention.

Figure 2 shows a flow chart showing an illustrative method for synthesizing a tracing agent of the invention.

Figures 3 and 4 are schematic representations of illustrative cassettes with associated columns and reagents for synthesizing a tracer agent of the invention using a modified GE TRACERlab-MX chemical module.

Figure 5 is a schematic representation of a system for synthesizing a tracer agent of the invention using a modified Explora GN chemical module.

Figure 6 shows an illustrative synthesis of a precursor of the tracer agent of the invention.

Figure 7 shows graphs of the percentage by weight versus time for the sulfuric salt of the tracer-1 precursor and the trifluoroacetic salt of the tracer-1 precursor.

Figures 8A-8F show HPLC chromatograms for compounds synthesized according to the methods described herein.

Figure 9A shows a graph illustrating changes in product distribution as a function of carbonate stoichiometry.

Figure 9B shows several by-products that can be formed during the synthesis of tracer-1 from the precursor of tracer-1.

Figure 9C shows a graph illustrating the changes in product distribution for the tracer-1 agent as a function of the stoichiometry of Et₄NHCO₃.

Figure 10 shows a graph illustrating the tissue distribution of the tracer-1 agent in mice suffering from tumors.

Other aspects, modalities and features of the invention will be apparent from the following detailed description, when considered in conjunction with the accompanying figures. The attached figures are schematic and are not intended to be drawn to scale. For clarification purposes, not all components in each figure are labeled, nor are all components of each embodiment of the invention shown, in cases where the illustration is not necessary for those skilled in the art to understand the invention. All patent applications and patents incorporated herein by reference are incorporated by reference in their entirety. In case of conflict, this description will prevail, including definitions.

Detailed description of the invention

The present invention relates generally to compounds, compositions thereof, systems comprising such compounds, reagents, cassettes, methods, kits and apparatus for the synthesis and / or use of tracer agents and precursors thereof. In some aspects, the invention generally relates to a tracer agent of the invention (ie, a tracer agent of formula (I), including a tracer agent of formula (V), such as a tracer agent-1) synthesized using methods described herein. The tracing agents of the invention can be used to acquire images of an area of ​​interest in a subject, including, without limitation, the heart, a portion of the heart, the cardiovascular system, the cardiac vessels, the brain and other organs.

In some embodiments, the present invention provides methods for synthesizing a precursor of the tracer agent of the invention that can be reacted with a tracer moiety (or a source thereof) to form a tracer agent. It is convenient to use methods that include high yield reactions and a relatively low number of synthetic, purification and / or formulation events in the preparation of a precursor of the tracer and / or tracer agent. Accordingly, many of the methods provided herein to synthesize a precursor of the tracer agent and / or the tracer agent provide the compounds in fewer steps than previously described, with a simpler synthesis and / or with a higher yield . In certain embodiments, the fluorination of a tracer agent precursor comprising a sulphonate type leaving group is carried out with a completely unprotected form of the precursor, which makes it unnecessary to employ a subsequent deprotection step. Therefore, the last synthetic step is the fluorination reaction, this prevents isotopically labeled material being lost in subsequent steps.

The methods and compositions of this description provide several advantages compared to the methods, compounds and compositions known in the art. As another example, some of the compounds provided herein are salts associated with a counter-anion, for which it has been found that, unexpectedly, the counter-anion improves solubility, yield, stability and / or ease of purification of the compound. For example, counter-anion in some cases affects numerous aspects of the manufacture of a tracer agent or a precursor thereof, or a composition thereof, including (1) the solubility of the precursor of the tracer agent and / or the tracer agent, ( 2) the purity of the tracer agent precursor and / or tracer agent, and (3) the stability of the tracer agent precursor and / or tracer agent.

Plotting agents

In some aspects, trace agents are provided to perform a tomography of an area of ​​interest of a subject. In certain embodiments, the tracer agent is marked with¹⁸F and is useful in PET tomography. In some embodiments, the tracing agent is a compound comprising the formula (I):

R⁴ NR²

K ^ Vn ^ N (R²)₂

R⁵

NR² or a salt, free base or combinations thereof, where:

R³, R⁴, R⁵ and R⁶ they can be identical or different and are individually hydrogen, Ci-C6 alkyl, heteroalkyl, halide, -0R⁷, -MR⁷, -N (R⁷) 2 or -C (= O) R⁸, each optionally substituted;

every R² it can be identical or different and is hydrogen or a nitrogen protecting group;

every R⁷ it may be identical or different and is hydrogen, alkyl, heteroalkyl, cycloalkyl, haloalkyl, heterocyclyl, aryl or heteroaryl, each optionally substituted;

every R⁸ it can be identical or different and is hydrogen, alkyl, heteroalkyl, cycloalkyl, haloalkyl, heterocyclyl, aryl, heteroaryl, -OH, alkoxy, -NH₂, alkylamino, -SH or alkylthiol, each optionally substituted;

m is an integer between 1 and 12, inclusive; and n is an integer between 1 and 4, inclusive.

In certain embodiments, the tracing agent is a compound comprising the formula (V):

<img file="MX367382B_D0034.tif" />

R⁶ (v) or a salt, free base or combinations thereof, where R³, R⁴, R⁵, R⁶, myn are as defined above.

In certain embodiments, the compound of formula (I) comprises the formula:

F

<img file="MX367382B_D0035.tif" />

where at least one R² It is a nitrogen protecting group.

In certain embodiments, the nitrogen protecting group is a Boc protecting group. In certain modalities, one, two or three R groups² they are nitrogen protecting groups (e.g., Boc protecting groups) and the other R groups² They are hydrogen. In certain embodiments, the fluoride of the compounds is isotopically enriched with^X8F. Fully protected, partially protected and completely unprotected forms of the compounds comprising formula (I) enriched with isotopically with ^XSF can be useful as tracing agents.

A non-limiting example of a tracer agent, referred to in the present tracer agent-1, comprises the formula:

NH

<img file="MX367382B_D0036.tif" />

The term "tracer-1 agent", as used herein, may also refer to a salt and / or a free base or combinations thereof of the above compound, such as a formate salt (Formula (VI)), a ascorbate salt (Formula (VII)), a citrate salt (Formula (IX)) or a trifluoroacetic salt (Formula (X)), as described herein.

For reasons of convenience and brevity, various aspects and embodiments of the invention are described in terms of the tracer-1 agent. However, it will be understood that, unless otherwise specified, the invention contemplates the synthesis and use of tracing agents different from the tracer-1 agent in these various aspects and modalities. Such tracing agents may be compounds of formula (I) and / or compounds of formula (V) as described herein.

The term "tracing agent", as used herein, refers to any chemical compound that includes a tracer moiety. A tracer moiety refers to an atom or group of atoms that is capable of producing by itself or by exposing it to an external energy source (e.g., electromagnetic radiation, ultrasound and the like) a detectable signal. The trace agents for use in nuclear medicine may comprise radioisotopes as tracer moieties. For example, trace agents for use in nuclear medicine may include ^{1: k}C, ¹³N, ¹⁸F, ⁷⁶Br, ¹²³I, ¹²⁴I, ^12SI, ¹³¹I, ^99mTc, ⁹⁵Tc, ^{11: L}In, ^S2Cu ⁶⁴Cu ^S7Ga and ⁶⁸Ga as tracer rest. In some modalities, the rest tracer is¹⁸F. Trace agents have been employed that comprise ¹⁸F to perform Pornographies of hypoxia and cancer (Drugs of the Future 2002, 27, 655-667).

The tracing agents allow to detect, acquire images and / or monitor the presence and / or progress of a condition, pathological disorder and / or disease. Typically, the tracer agent can be administered to the subject in order to obtain information related to at least a portion of the subject (eg, a human being). In some cases, a tracing agent can be used to highlight a specific area of ​​a subject, making organs, blood vessels, tissues and / or other portions more detectable and provide a clearer tomography. By increasing the detectability and / or image quality of the area to be studied, the presence and extent of the disease and / or injury can be determined.

In some embodiments, a tracing agent comprising an isotope, such as a radioisotope, may be referred to as isotopically enriched. An isotopically enriched composition refers to a composition comprising a percentage of one or more isotopes of an element that is greater than the natural percentage (of the isotope). For example, a composition that is isotopically enriched with a species of fluoride can be isotopically enriched with fluorine-18 (¹⁸F). Therefore, in relation to a plurality of compounds, when a particular atomic position is called¹⁸F, it should be understood that the abundance (or frequency) of ^ieF in that position (in the plurality) is superior, which includes substantially greater, than the natural abundance (or frequency) of ¹⁸F, which is essentially zero. In some embodiments, a fluorine called F may have a minimum isotopic enrichment factor of about 0.001% (i.e., about 1 in 10^s fluorine species is ^1SF), 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, approximately 0.05%, approximately 0.1%, approximately 0.2%, approximately 0.3%, approximately 0.4%, approximately 0.5%, approximately 0.75%, approximately 1%, approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 10%, approximately 15%, approximately 20%, approximately 30%, approximately 40%, approximately 50 %, approximately 60%, approximately 70%, about 80%, about 90%, about 95% or higher. The minimum isotopic enrichment factor, in some cases, can be between approximately 0.001% and approximately 1%. The isotopic enrichment of the compounds provided herein can be determined using conventional analytical methods with which one skilled in the art will be familiar, including mass spectrometry and HPLC.

In some embodiments, the methods and systems of this description employ or comprise compounds of formula (I) or (V), including, but not limited to, the tracer-1 agent. In some embodiments, the present invention relates to methods for performing a tomography, which include methods for performing a tomography on a subject that include administering a composition or formulation that includes a tracing agent (e.g. eg, a tracer agent comprising formula (I) or formula (V), such as tracer agent-1) to the subject by injection, infusion or any other known method, and performing a tomography of a region of the subject that be of interest Regions of interest may include, without limitation, the heart, a portion of the heart, the cardiovascular system, the cardiac vessels, the pancreas, the adrenal glands, the salivary glands, the thymus or other organs with a high sympathetic innervation or a high tracer agent absorption. Regions of interest may also include tumors. In certain embodiments, the tracer agent is used as a radiotracer for mapping cardiac nerve endings in vivo using positron emission tomography (PET) or other tomographic techniques. An event of interest can be tomographed and detected and / or other information can be determined using methods and / or systems of the description.

The tracing agents of the invention, including tracer-1 agent, can act as ligands of the norepinephrine transporter that have NET as a target or that bind to it. In some embodiments, the methods comprise detecting MET, including the determination of NET levels, in a subject, where the determination may comprise determining the level, density, function and / or location of NET in a subject. In certain embodiments, without wishing to adhere to any particular theory, the tracer agent binds to norepinephrine transporters (NET) to capture images of cardiac sympathetic activity or innervation. Accordingly, in some aspects, methods for assessing cardiac sympathetic innervation and / or myocardial sympathetic function are provided.

Plotter precursors

In other aspects, tracer agent precursors useful in the preparation of the tracer agents of the invention are provided. An illustrative synthesis of the precursor of the tracer-1 agent is shown in Figure 6. In certain embodiments, a precursor of the tracer agent of the invention comprises a leaving group (eg, a sulfonate) that can be substituted by a nucleophile in a substitution reaction. The tracer agent precursor may also include several functional groups that are optionally protected. The precursors of the synthesis of tracing agents of the invention are also contemplated by the present invention.

In certain embodiments, the present invention provides a compound (e.g., a precursor of the tracer agent) comprising the formula (II):

<img file="MX367382B_D0037.tif" />

(II), or a salt, free base or combinations thereof, where

R¹ it is alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, alkenyl, alkynyl or haloalkyl, each optionally substituted;

R³, R⁴, R⁵ and R⁶ they can be identical or different and are individually hydrogen, Ci-C6 alkyl, heteroalkyl, halide, -0R⁷, -MR⁷, -N (R⁷) 2 or -C (= O) R⁸, each optionally substituted;

every R² it can be identical or different and is hydrogen or a nitrogen protecting group;

every R⁷ it may be identical or different and is hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, haloalkyl, aryl or heteroaryl, each optionally substituted;

every R⁸ it can be identical or different and is hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, haloalkyl, aryl, heteroaryl, -OH, alkoxy, -nh₂, alkylamino, -SH or alkylthiol, each optionally substituted;

m is an integer between 1 and 12, inclusive; and n is an integer between 1 and 4, inclusive. In some embodiments, a compound of formula (II) is a precursor to the tracer.

In certain embodiments, the tracer agent precursor is a compound comprising formula (IV):

R1, 0; on

R⁵

NH

OR

NH₂ (IV) or a salt, free base or combination thereof, where R¹, R³-R⁶, myn are as defined herein.

A non-limiting example of a tracer agent precursor, referred to herein as the tracer agent-1, comprises the formula:

NH

OR

N NH,

H ² or a salt, free base or combinations thereof.

Another non-limiting example of a tracer agent precursor, referred to herein as the tracer-2 precursor, comprises the formula:

Br.

JÍ

NH

X

NH_?

H ² or a salt, free base or combinations thereof.

Another non-limiting example of a tracer precursor comprises the formula:

agent

NH

<img file="MX367382B_D0038.tif" />

or a salt, free base or combinations thereof.

Another non-limiting example of a tracer agent precursor comprises the formula:

NH

<img file="MX367382B_D0039.tif" />

or a salt, free base or combinations thereof.

Another non-limiting example of a tracer precursor comprises the formula:

agent

NBoc

<img file="MX367382B_D0040.tif" />

N NHBoc

H or a salt, free base or combinations thereof.

Another non-limiting example of a tracer precursor comprises the formula:

agent

NBoc

<img file="MX367382B_D0041.tif" />

or a salt, free base or combinations thereof.

Another non-limiting example of a tracer agent precursor comprises the formula:

NBoc

<img file="MX367382B_D0042.tif" />

NHBoc or a salt, free base or combinations thereof.

Another non-limiting example of a tracer agent precursor comprises the formula:

NBoc

<img file="MX367382B_D0043.tif" />

or a salt, free base or combinations thereof.

For reasons of convenience and brevity, various aspects and embodiments of the invention are described in terms of the precursor of the tracer-1 agent and / or the precursor of the tracer-2 agent. However, it will be understood that, unless otherwise specified, the invention contemplates the synthesis and use of precursors of tracing agents other than the precursor of tracer agent-1 and -2 in these various aspects and modalities. Such precursors of tracing agents may be compounds of formula (II) and / or compounds of formula (IV) and / or compounds of formula (III) as described herein.

In certain embodiments, a salt of a compound of formula (II) is provided. That is, a compound of formula (II) can be added and can be associated with a counterion. In some cases, the compound of formula (II) is positively charged. In a particular embodiment, the guanidine functional group of the compound of formula (II) is protonated and therefore positively charged such that a salt of a compound of formula (II) comprises formula (III):

<img file="MX367382B_D0044.tif" />

^r6 (III), where X® is a counter-anion. As those skilled in the art will understand, in embodiments described herein, in which a compound comprises a compound of formula (II), or a variation thereof, the compound may be present, at least in part, in a saline form . For example, any compound described herein comprising a neutral and / or deprotonated guanidine functional group may also be present as a protonated guanidine functional group (e.g. eg, associated with a counter-anion).

Those skilled in the art will be familiar with the appropriate counter-anions. In addition, those skilled in the art will know that the counter-anion Χθ can have a load greater than (-1) (e.g., (-2), (-3)), and in such modalities, each X® counter can be associated with more than one molecule of a compound of the present invention. Non-limiting examples of suitable counter-anions include the conjugated base of inorganic acids (e.g. e.g., chloride, bromide, iodide, fluoride, nitrate, sulfate, phosphate) or the conjugate base of organic acids (e.g., carboxylate, acetate, benzoate, tartrate, adipate, lactate, formate, maleate, glutamate, ascorbate, citrate, gluconate, oxalate, succinate, pamoate, salicylate, isethionate, succinamate, monodiglycolate, diisobutyrate, glucoheptonate). Other additional non-limiting examples of salts include adipate, alginate, aminosalicylate, anhydrometylene citrate, arecoline, aspartate, bisulfate, camforate, digluconate, dihydrobromide, disuccinate, glycerophosphate, hemisulfate, fluoride, iodide, methylenebis (salicylate, oxalate, oxalate, oxalate, oxalate, oxalate, oxalate, oxalate, oxalate, oxalate, oxalate, oxalate, oxalate, oxalate, oxalate , phenylethylbarbiturate, picrate, propionate, thiocyanate, tosylate, undecanoate, acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edentate, cansylate, carbonate, chloride, citrate, dihydrochloride, edentate, edisilate, stolate, silate, fumarate, gluceptate, gluconate, glutamate, glycolylarsanylate, hexylresorcinate, hydrabamino, bromide, chloride, hydroxynaphthoate, iodide, isethionate, lactate, lactate, lactate, lactate, malate mandelate, mesylate, mucate, napsilate, nitrate, pamoate (embonate), pantothenate, phosphate / diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tanate, tartrate, teoclate and triethiodide (see Berge et al., Journal of Pharmaceutical Sciences, 66 (1), 1977, 1-19). In certain embodiments, the salt is a mesylate (i.e. methanesulfonate), phosphate, sulfate, acetate, formate, benzoate, trifluoroacetate or tosylate salt of a compound of formula (II). In certain embodiments, the salt is a mesylate (ie, methanesulfonate), acetate, formate, benzoate, trifluoroacetate or tosylate salt of a compound of formula (II).

In some modalities, R¹ it is alkyl, haloalkyl or aryl. In some cases, R¹ it is alkyl (eg, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl). In some cases, R¹ is haloalkyl (e.g.

^and3 · '~ CF3, -CHF2, -CH2F, -CP2CF3, -CH2CF3). In some cases, R¹it is optionally substituted aryl. In certain modalities, R¹it is substituted or unsubstituted phenyl. In certain modalities, R¹ It is unsubstituted phenyl. In some cases, R¹ is substituted phenyl (e.g., 4-CH3Ph, 2,4,6 - (CH₃) ₃C₆H₂, C₆H, X where X is halide (e.g., 4-BrC₆H₄)).

<td>In some</td><td>modalities, n is</td><td>a</td><td>whole number</td>

<td>between</td><td>1 and 4, inclusive; or is it</td><td> 1, 2,</td><td>3 or 4</td>

<td>In some</td><td>modalities, m is</td><td>a</td><td>whole number</td>

<td>²⁵ between</td><td>1 and 12, inclusive, · or 1</td><td>and 10,</td><td>inclusive; or 1</td>

and 8, inclusive; or 1 and 6, inclusive; oesl, 2, 3, 4, 5o6. In some modalities, tn is an integer between 3 and 12, inclusive.

As described above, R² It can be a nitrogen protecting group. Nitrogen protecting groups are well known in the art and include those described in detail in Protecting Groups in Organic Synthesis, TW Greene and PGM Wuts, 3.^to edition, John Wiley &amp; Sons, 1999, which is incorporated herein by reference. For example, nitrogen protecting groups include, but are not limited to, carbamates (including methyl, ethyl and substituted ethyl carbamates (e.g., Troc), among others), amides, cyclic imide derivatives, N-alkyl - and N-arylamines, imine derivatives and enamine derivatives, among others. In some embodiments, the nitrogen protecting group is carbobenzyloxy (Cbz), p-methoxybenzylcarbonyl (MeOZ), t-butoxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc), acetyl (Ac), benzoyl (Bz), benzyl (Bn), pmethoxybenz (PMB), 3,4-dimethoxybenzyl (DMPM), pmethoxyphenyl (PMP) or p-toluenesulfonyloxy (Ts). In certain modalities, at least one R² it is t-butyloxycarbonyl (Boc).

Nitrogen protecting groups of the amide group type include, but are not limited to, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, Nbenzoylphenylaminoamide, benzylphenyl amide, acetamide, benzyl phenyl amide, acetamide o-nitrophenoxyacetamide, acetoacetamide, (N'-dithiobenzyloxycylamino) acetamide, 3 (p-hydroxyphenyl) propanamide, 3- (o-nitrophenyl) propanamide, 2-methyl-2- (o-nitrophenoxy) propanamide, 2-methyl1-2- (ophenylazophenoxy) propanamide, 4-chlorobutanamide, 3-methyl-3-nitrobutanamide, o-nitrocinamide, derivative of the type Nacethylmethionine, o-nitrobenzamide and (benzoyloxymethyl) benzamide.

The nitrogen protecting groups of the carbamate group type include, but are not limited to, methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9- (2-sulfo) fluorenylmethyl carbamate, 9- (2, 7-dibromo) fluoroenylmethyl, 2,7-di-t-butyl- [9- (10,10-dioxo-10,10,10,10 tetrahydrothioxantyl)] methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), carbamate of 2,2,2-trichloroethyl (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl (hZ) carbamate, 1- (1adamanti1) -1-methylethyl (Adpoc) carbamate, 1,1-dimethyl2-haloethyl carbamate, 1, l-dimethyl-2,2-dibromoethyl carbamate (DB- t-BOC), 1, l-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1-methyl-1- (4-biphenyl-1-ethyl) carbamate (Bpoc), 1- (3.5-carbamate) -di-t-butylphenyl) -1-methyl ethyl (tBumeoc), 2- (2 '~ and 4'-pyridyl) ethyl (Pyoc) carbamate, 2- (Ν, Ν-dicyclohexylcarboxamido) ethyl carbamate, t carbamate -butyl (BOC), 1-adamantyl carbamate (Adoc), Vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylalkyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinamyl carbamate (Noc), 8-quinolyl carbamate, Nhydroxypiperidinyl carbamate, alkyldithobamate , benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitrobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate (Ms 9-antrylmethyl carbamate, Diphenylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2- (ptoluenesulfoni1) ethyl carbamate, (2- (1,3-dithianyl)] methyl carbamate (Dmoc), 4-methylthiophenyl (Mtpc) carbamate, 2,4-dimethylthiophenyl (Bmpc), 2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphoniumphosphonium carbamate (Ppoc), 1,1-dimethyl-1-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate, p- ( dihydroxyboro1) benzyl, 5-benzisoxazolylmethyl carbamate, 2 (Trifluoromethyl) -6-Chromoniylmethyl Carbamate (Tcroc), m50 nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl (o-nitrophenyl carbamate ) methyl, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxyacylvinyl carbamate , o- (N, N-dimethylcarboxamido) benzyl carbamate, 1,1-dimethyl-3 (N, N-dimethylcarboxamido) propyl carbamate, 1,1-dimethylpropyl carbamate, di (2-pyridyl) methyl carbamate, 2-furanylmethyl carbamate , 2-iodoethyl carbamate, isobornyl carbamate, isobutyl carbamate, isonicotinyl carbamate, p- (p'-methoxyphenylazo) benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1-methyl-methylpropyl methylcarbamate , l-methyl-l- (3,5-dimethoxyphenyl) ethyl carbamate, l-methyl-l- (pphenylazophenyl) ethyl carbamate, 1-methyl-l-phenylethyl carbamate, ^ACd> 1-methi-1— (4-pyridyl) ethyl, phenyl carbamate, p- (phenylazo) benzyl carbamate, carbamate d

<td>2,4,6-tri-t-butylphenyl,</td><td>carbamate</td><td>from</td><td> 4-</td>

<td>(trimethylammonium) benzyl trimethylbenzyl.</td><td>and carbamate</td><td>from</td><td> 2,4,6-</td>

Sulfonamide group type nitrogen protecting groups include, but are not limited to, ptoluenesulfonamide (Ts), benzenesulfonamide, 2,3,6, trimethyl-4-methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6- dimethyl-4-methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte), 4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds , 2,2,5,7,8pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), β-trimethylsilylethanesulfonamide (SES), 9 anthracene sulfonamide, 4- (4 ', 8-dimethoxynaphthylmethyl) benzenesulfonamide (DNMBS), benzyl sulfonamide, trifluoromethyl sulfonamide and phenacylsulfonamide.

Other nitrogen protecting groups include, but are not limited to, a derivative of the phenythiazinyl- (10) acyl type, a derivative of the N'-p-toluenesulfonylaminoacyl type, a derivative of the N'-phenylaminothioacyl type, a derivative of the N-benzoylphenylalanyl type, N-derivative of acetylmethionine type,

4.5- dipheny1-3-oxazolin-2-one, N- £ thalimide, Ndithiasuccinimide (Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE) , 1,3-dimethyl-

1.3.5- 5-substituted triazacyclohexan-2-one, 1,3-dibenzyl-

1,3,5-t^r5-substituted iazacyclohexan-2-one, 1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N- [2 (trimethylsilyl) ethoxy] methylamine (SEM), N-3acetoxypropylamine, N- (l-Isopropyl-4-nitro-2-oxo-3-pyrrolin-3-yl) amine, quaternary ammonium salts, Nbenzylamine, N-di (4-methoxyphenyl) methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N - [(4methoxyphenyl) diphenylmethyl] amine (MMTr), N-9-phenylfluoroethylamine (PhF), N-2,7-dichloro-9-fluorenylmethyleneamine N-Ferrocenylmethylamino (Fcm), Ν-2-picolylamino N'oxide, Nl, l-dimethylthiomethylenamine, W-benzylidenamine, Np-methoxybenzylidenamine, N-diphenylmethylenamine, N - [(2-pyridyl) mesityl] methylenamine, N- (Ν , N '-dimethylaminomethylene) amine, N_t N'isopropylidenediamine, Np-nitrobenzylidenamine, Nsalicylidenamine, Ν-5-chlorosalicylidenamine, N- (5-chloro-2-hydroxyphenyl) phenylmethyleneamine, N-cyclohexylidenoamine, (5,5-dimethyl-3-oxo-l-cyclohexenyl) amine Nborane, derived from N-diphenylborinic acid, N [phenyl (pentaacrylicchromium- or tungsten) acyl] amine, N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, Ν'-amine oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, orobs ncsnosul f enarn i da (Nos) or δ-dini trobenzenesulfenamide, pentachlorobenzenesulfenamide,

2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide and 3-nitropyridinsulfenamide (Npys).

In some modalities, R⁴, R⁵ and R⁶ they are hydrogen; and R³it is Cx-Cg alkyl, Ch-Cg heteroalkyl, halide, -0R⁷, -MR⁷, N (R⁷)₂ or -C (= O) R⁸, each optionally substituted. In some cases, R³ is halo (e.g., F, C1, Br, I). In certain modalities, R³ It's bromine. In a particular mode, R⁴, R⁵ and R⁶ they are hydrogen; and R³ it is bromine, for example, so that the compound of formula (II) comprises the structure:

NR²

<img file="MX367382B_D0045.tif" />

In some modalities, each R² is hydrogen, so that the compound of formula (II) comprises the structure:

R⁴ NH

<img file="MX367382B_D0046.tif" />

R⁶

In a particular mode, R⁴, R^s and R⁶ they are hydrogen; R³ it is bromine; and every R² it is hydrogen, for example, so that the compound of formula (II) has the structure:

NH

In other modalities,

Br 0 „0 's' JA R'-

<img file="MX367382B_D0047.tif" />

NH₂

H at least one R² It is not hydrogen.

For example, the formulas:

the compound of

R⁴

<img file="MX367382B_D0048.tif" />

R⁶ formula (II) can be one of

NR²

NHR²

<img file="MX367382B_D0049.tif" />

NR²

R⁶

NH₂H

<img file="MX367382B_D0050.tif" />

NH

NR²

N ^ NHR²i H

R⁴

<img file="MX367382B_D0051.tif" />

R⁶

NH₂R²

R⁴

NH

<img file="MX367382B_D0052.tif" />

R⁶

<img file="MX367382B_D0053.tif" />

R⁶

As described herein, these compounds may be present as a salt, free base or combination thereof.

In some modalities, m is 3, n is 1, R³ is Br (or other halogen), and R⁴, R^s and R⁶ they are all H, so that a compound of formula (II) comprises the structure:

<img file="MX367382B_D0054.tif" />

NR²

OR

NN (R²)₂

R² where each of the R groups¹ and R² they are as defined above and as described in the modalities herein, both independently and in combination. In addition, in some cases, each R² is H, so that the compound of formula (II) comprises the structure:

NH

<img file="MX367382B_D0055.tif" />

where R¹ It is as defined above and as described in the modalities herein.

In certain embodiments, a compound of formula (II) comprises the structure:

NH

<img file="MX367382B_D0056.tif" />

or a salt, free base or combination thereof.

In certain embodiments, the present invention provides compounds useful in the synthesis of compounds of formula (II). In certain embodiments, the present invention provides a compound of the formula:

<img file="MX367382B_D0057.tif" />

or a salt, free base or combination thereof; where every R²it can be identical or different and is hydrogen or a nitrogen protecting group; and m is an integer between 3 and 12, inclusive. In one mode, m is 3.

In certain embodiments, the invention provides a compound comprising the formula:

NBoc

<img file="MX367382B_D0058.tif" />

or a salt, free base or combination thereof.

In certain embodiments, the invention provides a compound comprising the formula:

NR²

<img file="MX367382B_D0059.tif" />

or a salt, free base or combination thereof.

In one embodiment, the invention provides a compound comprising the formula:

NBoc

<img file="MX367382B_D0060.tif" />

<img file="MX367382B_D0061.tif" />

NHBoc or a salt, free base or combination thereof.

In one embodiment, the invention provides a compound comprising the formula:

NBoc

<img file="MX367382B_D0062.tif" />

or a salt, free base or combinations thereof.

In certain modalities, m is an integer between 3 and 10, inclusive; between 3 and 6, inclusive; or between 3 and 5, inclusive. In certain modalities, m is 3, 4, 5 or 6. In certain modalities, m is 3.

In some modalities, all R² are hydrogen In other modalities, at least one R² it is a nitrogen protecting group (e.g., nitrogen protecting groups described herein). In other modalities, at least two R² they are nitrogen protecting groups (e.g., nitrogen protecting groups described herein). In other modalities, at least three R² they are nitrogen protecting groups (e.g., nitrogen protecting groups described herein). In some embodiments, the nitrogen protecting group is carbobenzyloxy (Cbz), p-methoxybenzylcarbonyl (MeOZ), t-butyloxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc), acetyl (Ac), benzoyl (Bz), benzyl (Bn), p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP) or ptoluenesulfonyloxy (Ts). In certain modalities, at least one R² it is t-butyloxycarbonyl (Boc). In certain modalities, at least two R² they are t-butyloxycarbonyl (Boc).

In another aspect, the invention provides a compound comprising the formula:

<img file="MX367382B_D0063.tif" />

or a salt, free base or combination thereof, where m is an integer between 3 and 12, inclusive. In certain modalities, m is an integer between 3 and 10, inclusive; between 3 and 6, inclusive; or between 3 and 5, inclusive. In certain modalities, m is 3, 4, 5 or 6. In certain modalities, m is 3.

In one embodiment, the invention provides a compound comprising the formula:

<img file="MX367382B_D0064.tif" />

or a free base, salt or combination thereof.

In one aspect, the invention provides a compound comprising the formula:

<img file="MX367382B_D0065.tif" />

or a salt thereof, where m is an integer between 3 and 12, inclusive. In certain modalities, m is an integer between 3 and 10, inclusive; between 3 and 6, inclusive; or between 3 and 5, inclusive. In certain modalities, m is 3, 4, 5 or 6. In certain modalities, m is 3.

In one embodiment, the invention provides a compound comprising the formula:

<img file="MX367382B_D0066.tif" />

Methods of synthesis of precursors of tracing agents In other aspects, methods of synthesis of precursors of tracing agents of the invention and tracing agents of the invention are provided. In certain embodiments, a precursor of the tracing agent with a leaving group (e.g., sulfonate) is reacted with a nucleophile in a substitution reaction to obtain a tracing agent of the invention or a protected form thereof. Synthetic methods for preparing precursors prior to the synthesis of tracing agents of the invention are also provided, for example, synthetic methods whose illustrative steps are shown in Figure 6.

In some embodiments, the present invention provides methods for synthesizing precursors of tracing agents of the invention. The methods described herein can be used to synthesize a wide variety of tracer agent precursors. In general, the tracer agent precursor includes a leaving group that is replaced by a tracer moiety such as a kind of^youF.

The precursors of tracing agents of the invention (eg, compounds of formula (II)) can be prepared in several different ways. In certain embodiments, the free hydroxyl group of an alcohol comprising the formula (XI):

R⁴^R2R⁵ (XI), or a salt, free base or combinations thereof, where

R³, R⁴, R⁵ and R⁶ they can be identical or different and are individually hydrogen, Ci-Ce alkyl, heteroalkyl, halide, -OR, -SR⁷, -N (R⁷)₂ or -C (= O) R⁸, each optionally substituted;

every R² it can be identical or different and is hydrogen or a nitrogen protecting group;

every R⁷ it can be identical or different and is hydrogen, alkyl, heteroalkyl, cycloalkyl, halocyclyl heterocyclyl, aryl or heteroaryl, each optionally substituted;

every R⁸ it can be identical or different and is hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, haloalkyl, aryl, heteroaryl, -OH, alkoxy, -NH₂, alkylamino, -SH or alkylthiol, each optionally substituted;

m is an integer between 1 and 12, inclusive; and n is an integer between 1 and 4, inclusive, it is converted into a suitable leaving group (eg, a sulphonate leaving group) to obtain a compound comprising the formula (II). Each of the R groups²-R⁸, myn are as defined above and as described in the modalities herein, both independently and in combination, unless otherwise indicated. The methodology of the sulfonate leaving groups is reviewed in Netscher, Recent Res. Dev. Org. Chem. 7: 71-83, 2003, which is incorporated herein by reference. In certain embodiments, the free hydroxyl group is converted to a tosylate (4-methylbenzenesulfonate) using tosyl halide (e.g. eg, tosyl chloride). In certain embodiments, the free hydroxyl group is converted into a besylate (benzenesulfonate) using besylate halide (e.g._andj. , besylate chloride). In certain embodiments, the free hydroxyl group is converted to a nosylate (4-nitrobenzenesulfonate) using nosylate halide (eg, nosylate chloride). In other embodiments, the free hydroxyl group is converted to bromobenzenesulfonate using bromobenzenesulfonate halide (eg, bromobenzenesulfonate chloride). In other embodiments, the free hydroxyl group is converted to a mesylate (methanesulfonate) using mesyl halide (eg. , mesyl chloride). In other embodiments, the free hydroxyl group is converted into a triflate (trifluoromethanesulfonate) using triflic anhydride or a triflic halide. As one skilled in the art will appreciate, other sulfonates can be used in the precursors of tracing agents of the invention. Normally, the preparation of the sulfonate comprising the formula (II) is carried out in an aprotic solvent (e.g. , dichloromethane, THF) at room temperature or at a nearby temperature in the presence of a base such as a DMAP and / or a trialkylamine.

The alcohol comprising the formula (XI)_:

R⁴ NR² to ^r2R⁵ (XI) can be prepared based on synthetic methodology

<img file="MX367382B_D0067.tif" />

described in PCT Publication No. WO 2008/083056, which is incorporated herein by reference. In addition, Examples 1-13 and Figure 6 provide illustrative syntheses of several precursors of tracing agents of formula (II), including the salt forms thereof.

In certain embodiments, the alcohol comprising the formula (XI) is prepared by reacting a compound comprising the formula:

or a salt, free base or combination thereof, with a compound of the formula:

where LG is a suitable outgoing group. In one mode, m is 3.

In certain embodiments, the compound comprising the formula:

^{N (R2) J} It is of formula:

N

In one embodiment, the compound comprising the formula:

Ν 'is of formula:

In one embodiment, the compound comprising the formula:

It is of formula:

<img file="MX367382B_D0068.tif" />

In another aspect, the invention provides a method of preparing the starting material for the above reaction by reducing a compound comprising the formula:

<img file="MX367382B_D0069.tif" />

or a salt thereof, where m is an integer between 3 and 12, inclusive, with a reducer in suitable conditions to form a compound comprising:

<img file="MX367382B_D0070.tif" />

or a salt, free base or combination thereof. In one embodiment, m is 3. Illustrative agents useful in reducing a nitrile group (-CN) to obtain a primary amino group (-CH₂NH₂), include, without limitation

LiAlH, (LAH)_; gaseous hydrogen (H₂) in the presence of a metal catalyst (e.g., Pd, Pt, Ni); NaBH₄ and a salt of a transition metal to form the metal borate in situ (e.g., NiCl₂ to form nickel borate (NiBH₄) in situ; ZnCl₂ to form zinc borate (ZnBH₄) in situ); NaBH₄ together with I₂; NaBH, together with H₂SW₄; NiBH₄; ZnBH₄; LiBH₄; and borane (e.g., BHj / THF, BH₃/ DCM). In one embodiment, the reducer is borane (e.g., BH₃/ THF).

In some embodiments, the invention provides a method for deprotecting a guanidine functional group of a compound comprising formula (II):

R⁴ NR²R³ JL

R¹'^S'O' ^ O '^^xf'R^{5 R2 r6} (II).

For example, in some embodiments, a method comprises deprotecting a guanidine functional group of a compound comprising formula (II):

R⁴ NR²

<img file="MX367382B_D0071.tif" />

R® (II) or a salt, free base or combination thereof, under suitable conditions to form a compound comprising the formula (IV):

<img file="MX367382B_D0072.tif" />

R® (IV), or a salt, free base or combination thereof, where

R¹ it is alkyl, heteroalkyl, cycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, alkenyl, alkynyl, heterocyclyl or haloalkyl, each optionally substituted;

R³, R⁴, R^s and R⁶ they can be identical or different and are individually hydrogen, CA-Cs alkyl, heteroalkyl, halide, -0R⁷, -MR⁷, -N (R⁷)₂ or -C (= O) R⁸, each optionally substituted;

every R² it can be identical or different and is hydrogen or a nitrogen protecting group;

every R⁷ it can be identical or different and is hydrogen, alkyl, heteroalkyl, cycloalkyl, haloalkyl, aryl, heteroaryl or heterocyclyl, each optionally ¹⁵ replaced, each R⁸ it can be identical or different and is hydrogen, alkyl, heteroalkyl, cycloalkyl, haloalkyl, heterocyclyl, aryl, heteroaryl, -OH, alkoxy, —NH₂, alkylamino, -SH or alkylthiol, each optionally substituted;

<td colspan="2">m is a</td><td rowspan="2">number</td><td rowspan="2">whole</td><td rowspan="2">understood</td><td rowspan="2">between 1 and</td><td rowspan="2"> 12,</td>

<td></td><td>inclusive; Y</td>

<td></td><td>n is an inclusive.</td><td>number</td><td>whole</td><td>understood</td><td>between 1 and</td><td> 4,</td>

<td> 25</td><td>Each</td><td>of the</td><td>groups</td><td>R^x-R⁸my</td><td>n are like</td><td>I know</td>

defined above and as described in the modalities herein, both independently and in combination, unless otherwise indicated.

Suitable conditions for deprotecting a guanidine functional group are described herein. Such conditions may include an acidic environment (eg, pH less than or equal to 4, less than or equal to 3, less than or equal to 2, or less than or equal to 1). For example, in certain modalities, one or more R groups² they are t-butyloxycarbonyl and the deprotection step comprises treating a compound of formula (II) with trifluoroacetic acid, hydrochloric acid, sulfuric acid or p-toluenesulfonic acid. Such deprotection conditions may include, in addition or as an alternative, a temperature between 100-150 ° C.

The methods described herein can be carried out in any suitable solvent including, but not limited to, non-halogenated hydrocarbon solvents (e.g., pentane, hexane, heptane, cyclohexane), halogenated hydrocarbon solvents (e.g., dichloromethane , chloroform, fluorobenzene, trifluoromethylbenzene), aromatic hydrocarbon solvents (e.g., toluene, benzene, xylene), ester type solvents (e.g., ethyl acetate), ether type solvents (e.g. ex. , tetrahydrofuran, dioxane, diethyl ether, dimethoxyethane) and alcohol solvents (eg, ethanol, methanol propanol, isopropanol, tert-butanol). In certain embodiments, a protic solvent is used. In other embodiments, an aprotic solvent is used. Non-limiting examples of useful solvents include acetone, acetic acid, formic acid, dimethyl sulfoxide, dimethylformamide, acetonitrile, p-cresol, glycol, petroleum ether, carbon tetrachloride, hexamethylphosphoric triamide, triethylamine, picoline and pyridine.

The methods can be carried out at any suitable temperature. In some cases, the method is carried out at about room temperature (eg, about 20 'C, between about 20 ° C and about 25' C, about 25 'C or similar). However, in some cases, the method is carried out at a temperature below or above room temperature, for example, at about 78 'C, at about -70' C, about -50 'C, about -30' C, about -10 ° C, about -0 'C, about 10' C, about 30 'C, about 40' C, about 50 'C, about 60' C, about 70 'C, about 80' C, about 90 'C, approximately 100' C, approximately 120 'C, approximately 140' C or the like. In some embodiments, the method is carried out at temperatures above room temperature, for example, between

<td>approximately</td><td> 25</td><td>° C</td><td>Y</td><td>approximately</td><td> 120</td><td>° c,</td><td>or</td><td>between</td>

<td>approximately</td><td> 25</td><td>° C</td><td>Y</td><td>approximately</td><td> 100</td><td>° c,</td><td>or</td><td>between</td>

<td>approximately</td><td> 40</td><td>° C</td><td>Y</td><td>approximately</td><td> 120</td><td>° C,</td><td>or</td><td>between</td>

<td>approximately</td><td> 80 '</td><td>° C and</td><td colspan="2">about 120</td><td>° C.</td><td colspan="3">Temperature</td>

It can be maintained by refluxing the solution. In some cases, the method is carried out at temperatures between -78 ° C and approximately 25 ° C, or between approximately 0 "C and approximately 25 'C.

The methods described herein can be carried out at any suitable pH, for example, less than or equal to about 13, less than or equal to about 12, less than or equal to about 11, less than or equal to about 10, less than or equal to about 9, less than or equal to about 8, less than or equal to about 7, or less than or equal to about 6. In some cases, the pH may be greater than or equal to 1, greater than or equal to 2, greater than or equal to 3, greater than or equal to 4, greater than or equal to 5, greater than or equal to 6, greater than or equal to 7, or greater than or equal to 8. In some cases, the pH may be between about 2 and

<td>approximately</td><td> 12,</td><td>OR</td><td>between</td><td>approximately</td><td> 3</td><td>Y</td>

<td>approximately</td><td> 11,</td><td>or</td><td>between</td><td>approximately</td><td> 4</td><td>Y</td>

<td>approximately</td><td> 10,</td><td>or</td><td>between</td><td>approximately</td><td> 5</td><td>Y</td>

<td>approximately</td><td> 9,</td><td>or</td><td>between</td><td>approximately</td><td> 6</td><td>Y</td>

about 8, or about 7.

The yield percentage of a product may be greater than about 60%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90% , greater than about 5 92%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, greater than about 99% or higher.

Synthesis methods of trace agents ¹⁰ In other aspects, methods for synthesizing plotting agents are provided. The methods described herein can be used to synthesize various tracing agents of the invention from a precursor of the tracing agent of the invention.

Fluoridation

In some cases, the tracer agent is formed by reacting a precursor to the tracer agent (eg, a compound comprising formula (II) - (IV)) with a tracer moiety. The tracer agent precursor may include at least one leaving group that is susceptible to being displaced by a nucleophilic tracer moiety such as a fluoride species¹⁸F. Therefore, in certain embodiments, the method involves reacting a precursor of the tracer agent comprising a leaving group with a source of a moiety ²⁵ tracer (e.g., a kind of fluoride). For example, during the reaction, the tracer moiety replaces the leaving group by a substitution reaction such as an S-type reaction._None or S_N2, in this way the tracer agent is produced. In certain embodiments, the fluorination reaction is a single step procedure that does not require a subsequent deprotection step. That is, the fluorination step is carried out in a precursor of the completely unprotected tracer. A non-limiting example of a synthetic method for preparing a tracer agent is shown in Figure 1, where the precursor of tracer agent-1 becomes tracer agent-1. In some embodiments, multiple substitution reactions may occur across multiple leaving groups during the synthesis of a tracer from a tracer agent precursor. The methods described herein have improved yields and may allow the synthesis of tracer agents, including tracer agents comprising a radioisotope (e.g.,^iaF). Plotting agents can be useful as sensors, diagnostic tools and the like. Synthetic methods have also been designed to prepare a tracer agent that uses an automated synthesis system to prepare and purify tracer agents comprising a radioisotope.

As described herein, in some cases, the method of synthesis of a tracing agent of the invention may involve the use of one or more reagents (e.g., salts) that may favor a chemical reaction (e.g. ., a substitution reaction). In certain embodiments, the selection of a salt form may allow the fluorination of a precursor of the unprotected tracer agent. Without wishing to adhere to any particular theory, counter-anion can interact with the guanidine functional group, which prevents it from interfering with the fluorination reaction and / or can prevent secondary reactions. In certain embodiments, the salt is a mesylate (ie, methanesulfonate), phosphate, sulfate, acetate, formate, benzoate, trifluoroacetate or tosylate salt of a compound of formula (II). In certain embodiments, the salt is a mesylate salt. (i.e. methanesulfonate), acetate, formate, benzoate, tififluoroacetate or tosylate of a compound of formula (II)

In some embodiments, a method of synthesizing a tracing agent comprises contacting a precursor of the tracing agent of the invention (e.g., a compound comprising the formula (II), (m) _or (χ_ν)) _with a species of fluoride, which causes the fluoride species to replace the leaving group of the precursor to produce a tracing agent (e.g., a compound comprising formula (I)) comprising the fluoride species

In certain embodiments, the method involves a nucleophilic fluorination reaction. That is, the precursor of the tracer agent comprising a leaving group is reacted in the presence of a fluoride species, whereby an S-type shift_None or S_Ntwo from the leaving group by the fluoride species produces the tracing agent. In some embodiments, the fluoride species is isotopically enriched with¹⁸F.

Those skilled in the art will be familiar with the conditions suitable for fluorination of a compound (eg, a compound of formula (II), (III) or (IV)). For example, refer to International Patent Application No. PCT / US2011 / 024109, of Cesati, filed on February 8, 2011, incorporated herein by reference. In some cases, a compound of formula (II), (III) or (IV), or a salt, free base or combination thereof, is exposed to a fluorine source, optionally enriched with a fluorine isotope (e.g. . , enriched with ^ieF). In some cases, the source of fluoride is a fluoride salt (e.g., KF, NaF, tetralkylammonium fluoride).

The fluorine source may comprise or be associated with another reagent, or it may be used in conjunction with it. The reagent may be able to increase the reactivity of the fluorine species or to favor the conversion of the precursor θη the tracing agent. For example, in a group of embodiments, the reagent can be used in combination with a multidentate ligand, such as a crown ether or a crypto, which is capable of chelating a metal ion. The multidentate ligand can be, for example, 4,7,13,16,21,24-h.exaoxa1,10-diazabicyclo [8.8.8] hexacosan (ie, Kryptofix® 222). When the source of fluorine is KF, cryptands with a high affinity for potassium are useful since they chelate potassium and thus increase the reactivity of the fluoride ion. In some embodiments, cryptands with a potassium affinity similar to that of Kryptofix® 222 are used (e.g. 75%, 80%, 85%, 90%, 95% or a higher percentage of the affinity of Kryptofix 222 for potassium). The reaction conditions may comprise one or more solvents.

In some embodiments, fluorination occurs in the presence of K2CO3 and Kryptofix 222 (or any other crypto with an affinity for the cation of interest, including, for example, potassium, similar to that of Kryptofix 222) in MeCN (acetonitrile) alone. or combined with t-BuOH, as solvent. The molar ratio of K₂CO₃ with respect to the tracer agent precursor (such as, without limitation, the tracer agent precursor-1 or -2) is between about 0.5: 1 and about 5: 1, more preferably between 0.5: 1 and 1: 1. In some modalities, the molar ratio is approximately

0.66:1.

In some embodiments, fluorination occurs in the presence of tetraalkylammonium carbonate or tetraalkylammonium bicarbonate in MeCN as solvent. In some embodiments, the molar ratio of tetraalkylammonium carbonate or bicarbonate to the precursor of the tracer (such as the precursor of the tracer-1 or -2) is 5: 1. In some embodiments, the molar ratio may be between about 7: 1 and about 3: 1, or between about 6: 1 and about 4: 1, or between about 5.5: 1 and about 4.5: 1. The tetraalkylammonium cation may be tetraethylammonium or tetrabutylammonium but is not limited to these.

Compounds comprising the formula (V):

R⁴ NH

<img file="MX367382B_D0073.tif" />

or a salt, free base or combination thereof, where each of the R groups³-R⁶, myn are as defined above and as described in the embodiments herein, both individually and in combination, can be produced from a precursor using a two step or three step process as described in the Publication of PCT International WO 2008/083056 of Purohit et al., which is incorporated herein by reference.

In contrast, the synthetic methods provided herein may involve the single-step preparation of tracing agents of the invention (eg, compounds of formula (V), or a salt, free base or combination thereof). The one-step method involves minimal fluoridation of a completely or partially unprotected precursor in the presence of, for example, K₂C0₃/ Kryptofix® 222 (or other suitable alternatives to Kryptofix® 222), or tetraalkylammonium carbonate or bicarbonate, in MeCN alone or mixed with MeCN (such as a mixture of MeCN and tBuOH). These methods are particularly suitable when particular salt forms of the precursors of tracing agents of the invention are used. These salts include halide, acetate, formate, citrate, ascorbate, trifluoroacetate, toluenesulfonate, benzoate, acetate, phosphate, sulfate, tosylate and mesylate.

In some cases, the methods also identify important counterions in the production of salts of a compound of formula (V). In some cases, the counter-anion can affect: (1) the solubility of the precursor, (2) the purity of the active pharmaceutical intermediate and (3) the stability of the pharmaceutical product. In some cases, the trifluoroacetate anion proved to be particularly effective. In certain embodiments, such as those described herein, the precursor of the tracing agent and / or the tracing agent are present in a saline form that favors the reactivity and / or stability of the reaction product and / or reagent during and / or after a deprotection and / or fluorination reaction.

In some cases, the tracer agent precursor comprises a guanidine functional group that may or may not be unprotected before or, in some cases, after fluorination. For example, the guanidine functional group of a compound of formula (II) may or may not be unprotected before fluorination. That is, in some cases, a tracer agent precursor comprising a protected guanidine functional group is subjected to fluorination and optionally subsequent deprotection. Alternatively, the guanidine functional group of a tracer agent precursor is deprotected (eg, according to the methods described herein) and then subjected to fluorination. As described herein, in certain embodiments, the fluorine source is isotopically enriched with¹⁸F.

In certain embodiments, a compound comprising formula (II) is first subjected to fluorination and then deprotected. In certain embodiments, the method comprises fluoridation of a compound comprising formula (II):

R⁴ NR² r3 Jf Μ 11 0 0 YVb NN (R²)₂^R2^r6 (II) or a salt, free base or combination thereof, under suitable conditions to form a compound comprising the formula (I):

R⁴ NR²r3 ϊ ΙΛ XV ^ Í^&lt;Vn ^ 'N (R²)₂m J! p2^R'(i).

or a salt, free base or combination thereof, where

R³, R⁴, R⁵ and R⁶ they can be identical or different and are individually hydrogen, Cj-Cg alkyl, heteroalkyl, halide, —OR, -SR, -N (R⁷)₂ or -C (= O) R⁸, each optionally substituted;

every R² it can be identical or different and is hydrogen or a nitrogen protecting group;

every R⁷ it may be identical or different and is hydrogen, alkyl, heteroalkyl, cycloalkyl, haloalkyl, aryl, heteroaryl or heterocyclyl, each optionally substituted;

every R⁸ it can be identical or different and is hydrogen, alkyl, heteroalkyl, cycloalkyl, haloalkyl, heterocyclyl, aryl, heteroaryl, -OH, alkoxy, -NH_Z, alkylamino, -SH or alkylthiol, each optionally substituted;

m is an integer between 1 and 12, inclusive; and n is an integer between 1 and 4, inclusive.

Each of the R groups¹-R^fi, myn are as defined above and as described in the modalities herein, both independently and in combination, unless otherwise indicated.

Suitable conditions for the fluorination of a compound are described herein.

In some cases, after fluoridation of a compound comprising formula (II) to form a compound comprising formula (I), the compound comprising formula (I) is completely or partially deprotected. In certain embodiments, the method comprises deprotecting the compound comprising the formula (I):

R⁴

NR²^ N ^ N (R²)₂

N

R²

R⁶ or a salt, free base or combination thereof, provided that at least one R is not H, under suitable conditions to form a compound comprising the formula (V):

R⁴ NH

<img file="MX367382B_D0074.tif" />

or a salt, free base or combination thereof. Deprotection can be carried out, for example, under acidic conditions (eg, pH less than or equal to 4) and optionally at elevated temperatures (eg, between 100 and 150 ° C).

However, in some cases, a tracer agent precursor comprising an unprotected guanidine functional group is fluorinated. For example, in certain embodiments, the method comprises the fluorination of a compound comprising the formula (IV):

R⁴ NH

<img file="MX367382B_D0075.tif" />

^R6 (IV) or a salt, free base or combination thereof, under suitable conditions to form a compound of formula (V):

<img file="MX367382B_D0076.tif" />

or a salt, free base or combination thereof, where it is alkyl, heteroalkyl, cycloalkyl aryl, heteroaryl, arylalkyl, heteroarylalkyl, alkenyl, alkynyl, heterocyclyl or haloalkyl, each optionally substituted;

R³, R⁴, R⁵ and R⁶ they can be identical or different and are individually hydrogen, Cx-Cg alkyl, heteroalkyl, halide, -0R⁷, -MR⁷, -N (R⁷)₂ or -C (= O) R⁸, each optionally substituted;

every R⁷ it may be identical or different and is hydrogen, alkyl, heteroalkyl, cycloalkyl, haloalkyl, aryl, heteroaryl or heterocyclyl, each optionally substituted;

every R⁸ it can be identical or different and is hydrogen, alkyl, heteroalkyl, cycloalkyl, haloalkyl, heterocyclyl, aryl, heteroaryl, -OH, alkoxy, -NH₂, alkylamino, -SH or alkylthiol, each optionally substituted;

m is an integer between 1 and 12, inclusive; and n is an integer between 1 and 4, inclusive.

Each of the R groups¹r³-r⁸, _m and n are as defined above and as described in the modalities herein, both independently and in combination, unless otherwise indicated.

In some cases, it has been found that the solubility and / or reactivity stability of an ester of the precursor sulfonic acid against that of its fluorinated counterpart depends on the derived guanidinium salt form. For example, an investigation of a series of salts of mineral acids (eg, salts of chloride, phosphate and sulfate) demonstrated variable physical properties relevant to long-term manufacturing and storage capacity. The development of salt forms revealed differences in solubility in multiple solvent systems relevant to modern fluorination chemistry including, for example, MeCN, t-BuOH and mixtures thereof. In some cases, the solubility of the agent precursor was correlated with the overall fluorination efficiency, since minimum concentration thresholds of the tracer agent precursor were required to achieve preferential fluorination rates relative to decomposition. In addition, in some cases, the reaction rates also varied with the counter-anion selection, even for equivalent molarity values ​​of the solution.

In some embodiments, a method of synthesizing a fluorinated compound comprises reacting, in the presence of a reagent (e.g., a carbonate or bicarbonate ion), (i) ^a precursor of the fluorinated compound comprising a substituent substituted with a halide or a group containing sulfate onato, with (ii) a salt comprising a species of fluoride and a weakly coordinating cation.

The term "leaving group", as used herein, is given its usual meaning in the field of synthetic organic chemistry and refers to an atom or a group that can be displaced by a nucleophile. Examples of suitable leaving groups include, but are not limited to, halides (such as chloride, bromide or iodide), alkoxycarbonyloxy, aryloxycarbonyloxy, alkanesulfonyloxy, arenesulfonyloxy, alkylcarbonyloxy (e.g. eg, acetoxy), arylcarbonyloxy, aryloxy, methoxy, N, O-dimethylhydroxylamino, ρϊ^χϊ1 ° &lt;and haloformates. In some cases, the leaving group is an ester of sulfonic acid, such as toluenesulfonate (tosylate, Ts), methanesulfonate (mesylate, Ms), pbromobenzenesulfonyl (brosylate, Bs) or trifluoromethanesulfonate (triflate, Tf). In some cases, the leaving group may be a brosylate such as pbromobenzenesulfonyl. In some cases, the leaving group may be a nosylate such as 2-nitrobenzenesulfonyl. The leaving group can also be a phosphinoxide (e.g. , formed during a Mitsunobu reaction) or an internal leaving group such as an epoxy or a cyclic sulfate. In some embodiments, the leaving group is a group containing sulfonate. In some embodiments, the leaving group is a tosylate group.

In some embodiments, one or more reagents are used in the reaction mixture comprising the tracer agent precursor and the fluoride species. A reagent, also called an additive, refers to any chemical compound added to the reaction mixture. The reagent may or may not be consumed during the reaction. The reagent can be a stoichiometric or catalytic reagent. Illustrative reagents include catalysts, salts, oxidants, reducing agents, chelating agents, bases, acids, metals, phase transfer reagents and others, as one skilled in the art will appreciate.

The reagent may, in some cases, favor the reaction between the tracer agent precursor and the fluoride species, and / or may contribute to the stabilization of the resulting tracer agent. For example, the fluoride species could have a relatively low reactivity (eg, nucleophilia) and the addition of certain reagents could increase the reactivity of the fluoride species. By way of illustration, a kind of fluoride could be a negatively charged fluoride ion (e.g., an ion ¹⁸P isotopically enriched) and a reagent could be used to bind any positively charged counterions that are present in the reaction mixture, thereby increasing the reactivity of the fluoride ion. An example of this type of reagent is a crypto, such as, without limitation, Kryptofix (eg, Kryptofix®-222). In some embodiments, the reagent reduces the rate of unwanted side reactions, as described below.

In some cases, the reagent may be combined with the fluoride species before contacting it with the tracer agent precursor. For example, in certain embodiments, a solution comprising the fluoride species and the reagent is prepared, and the solution is added to the tracer agent precursor. In other embodiments, a solid comprising the fluoride species and the reagent is prepared, and the solid is contacted with the precursor of the tracing agent in solution. In certain embodiments, the fluoride species is adsorbed on a solid support (eg, an anion exchange column) and a solution comprising the reagent is used to elute the fluoride species from the solid support. Then, the eluted solution is contacted with the tracer agent precursor or concentrated to produce a solid, which is subsequently contacted with the tracer agent precursor in solution.

In some embodiments, the reagent is a bicarbonate salt. The term "bicarbonate salt," as used herein, refers to a salt comprising a bicarbonate or hydrogen carbonate ion (HCÓ ion₃ ). The bicarbonate salt can be a bicarbonate of a metal such as sodium bicarbonate, calcium bicarbonate, potassium bicarbonate and magnesium bicarbonate. In certain embodiments, the bicarbonate salt is potassium bicarbonate (KHCO₃). In some embodiments, the bicarbonate salt comprises a non-metallic counterion such as ammonium bicarbonate. For example, the bicarbonate salt may be a tetraalkylammonium bicarbonate salt with the formula R₄NHCO₃where R₄ It is alkyl. In some embodiments, R may be a lower alkyl such as methyl, ethyl, propyl, butyl, pentyl, hexyl or the like. In certain modalities, ammonium salt¹⁰ it's Et₄NHCO₃. In other modalities, salt is Me₄NHCO₃, iPr₄NHCO₃, n Pr₄NHCO₃, n-Bu₄NHCO₃, i-Bu₄NHCO₃ or t-Bu₄NHCO₃.

In some embodiments, the reagent is a carbonate salt. The term "carbonate salt", as used herein, refers to a salt comprising a carbonate ion (C0 ion₃’²). The carbonate salt can be a carbonate of a metal such as sodium carbonate, calcium carbonate, potassium carbonate and magnesium carbonate. In certain embodiments, the carbonate salt is potassium carbonate (K₂CO₃). In some embodiments, the 2 0 carbonate salt comprises a non-metallic counterion such as ammonium carbonate. For example, the carbonate salt may be a tetraalkylammonium carbonate salt with the formula (R₄N)₂CO₃, where R is alkyl. In some embodiments, R may be a lower alkyl such as methyl, ethyl, propyl, butyl, pentyl, hexyl or the like. In certain embodiments, the ammonium salt is (Et₄N)₂CO₃. In other modalities, salt is (Me₄N)₂CO₃, (i-Pr₄N)₂CO₃, (n-Pr₄N) ₂CO₃, (n-Bu₄N) ₂CO₃, (i-Bu₄N)₂CO₃o (t-Bu₄N) ₂CO₃.

Without wishing to adhere to any particular theory, the use of bicarbonate, carbonate and / or ammonium salts can contribute to the reduction of the speed of competitive reactions, such as hydrolysis, during the nucleophilic fluorination of a tracer agent precursor.

In some embodiments, the reagent is a salt comprising a cation that forms a weakly coordinating salt with a kind of fluoride. The term "cation that forms a weakly coordinating salt with a species of fluoride, as used herein, refers to a cation that provides a species of reactive fluoride in a fluorination reaction. For example, the cation may not bind strongly to the fluoride species, which allows the fluoride species to act as a nucleophile during a nucleophilic fluorination reaction. Those skilled in the art will be able to select an appropriate cation that is suitable as a weakly coordinating counterion for a fluoride species. For example, the cation may have a relatively large atomic radius and / or may be a weak Lewis base. In some cases, the cation can be selected to be lipophilic. In some cases, the cation may comprise one or more alkyl groups. Examples of weakly coordinating cations include cesium ions, ammonium ions, weakly coordinating salts of hexamethylpiperidinedium, S (NMe₂)₃, P (NMe₂)₄, tetraaalkylphosphonium salts, tetraarylphosphonium salts, (e.g., tetraphenylphosphonium), hexakis (dimethylamino) diphosphazenium and tris (dimethylamino) sulfonium.

In some embodiments, the reagent is an ammonium salt, that is, a salt comprising a substituted or unsubstituted ammonium ion. In some cases, the ammonium ion is a weakly coordinating cation. In some cases, the ammonium salt has the formula R₄NX, where each R may be the same or different and is alkyl, heteroalkyl, aryl, heteroaryl or heterocyclyl, each optionally substituted, and X is a negatively charged counterion. In some cases, R is alkyl, heteroalkyl, aryl, heteroaryl or heterocyclyl, each optionally substituted. The ammonium salt may include a range of negatively charged counterions, which include halides, carbonates and bicarbonates. Examples of ammonium salts include, but are not limited to, ammonium bicarbonate type salts, ammonium hydroxide type salts, ammonium acetate type salts, ammonium lactate type salts, ammonium trifluoroacetate type salts, type salts ammonium methanesulfonate, ammonium nitrate p-toluenesulfonate type salts, ammonium nitrate type salts, ammonium halide type salts (eg, ammonium iodide type salts) and ammonium bisulfate type salts.

In a set of embodiments, the ammonium salt is a tetraalkylammonium salt such as a tetraalkylammonium bicarbonate salt. For example, the ammonium salt can have the formula R₄NHCO₃, where each R is independently alkyl. In some cases, R is optionally substituted. In some embodiments, the alkyl group is a lower Ci-Cg alkyl group. In some embodiments, the tetraalkylammonium salt is a basic tetraalkylammonium salt.

The salt (p. eg, bicarbonate and / or ammonium salt) can be used in the reaction so that the molar ratio of the salt to the precursor of the tracer is less than or equal to about 10: 1, or less than or equal to about 9: 1, or less than or equal to about 8: 1, or less than or equal to about 7: 1, or less than or equal to about 6: 1, or less than or equal to about 5: 1, or less than or equal to about 4: 1, or less than or equal to about 3: 1, or less than or equal to about 2: 1, or less than or equal to about 1: 1. In some cases, the

<td>molar ratio</td><td colspan="2">of salt</td><td>respect</td><td colspan="3">to the agent precursor</td>

<td>plotter is</td><td colspan="2">understood</td><td>between</td><td>approximately</td><td> 3 :1</td><td>Y</td>

<td>approximately</td><td> 8:1,</td><td>OR</td><td>between</td><td>approximately</td><td> 4 : 1</td><td>Y</td>

<td>approximately</td><td> 7:1,</td><td>or</td><td>between</td><td>approximately</td><td> 5 : 1</td><td>Y</td>

<td>approximately</td><td> 7:1,</td><td>or</td><td>between</td><td>approximately</td><td> 5 : 1</td><td>Y</td>

approximately 8: 1.

In some embodiments, the reagent is used in combination with a species capable of increasing the reactivity of the fluoride species or that favors the conversion of the tracer agent precursor into the tracer agent. For example, the species can be a compound capable of chelating one or more ions (e.g. eg, metal ions) that are present in the reaction mixture, without wishing to adhere to any theory, the species can be used to chelate a counterion to a species of fluoride, such as a potassium ion, to thereby increase the reactivity (e.g., nucleophilia) of the fluoride species. In certain embodiments, the reagent is used in combination with a multidentate ligand, such as a crown ether or a crypto, which is capable of chelating a metal ion. The multidentate ligand (e.g. ex. , a crypto) can be selected based on the metal ion to be chelated. The multidentate ligand can be, for example, 4,7,13,16,21,24-hexaoxa-1,10diazabicyclo [8.8.8] hexacosan (eg, Kryptofix® 222). Those skilled in the art will know other cryptands.

Some modalities involve the use of a carbonate salt combined with 4,7,13,16,21,24-hexaoxa-1,10diazabicyclo [8.8.8] hexacosan. In a specific embodiment, potassium carbonate combined with 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo [8.8.8] hexacosan is used.

In another group of modalities, it may be beneficial to employ the methods described herein in the absence of a crypto. The term crypto is given its usual meaning in the art and refers to a bi- or polycyclic multidentate ligand for a cation. For example, the method can be carried out using an ammonium salt, in the absence of a crypto (eg, 4,7,13,16,21,24-hexaoxa1,10-diazabicyclo [8.8.8] hexacosan ). In some cases, cryptands may increase the pH of the reaction solution, which in the presence of another reagent (e.g., carbonate salt) may adversely affect the yield and / or purity of the fluorination reaction. Therefore, if the fluorination reaction is carried out in the absence of a crypto and optionally in the presence of another reagent (e.g. eg, bicarbonate and / or ammonium salt), the yield and / or the purity of the reaction can be increased, as described herein.

In another group of modalities, the method is carried out in the absence of a carbonate salt.

<td>In some</td><td colspan="2">modalities, the</td><td>use of a salt in</td><td colspan="2">the reaction</td>

<td>increase the</td><td colspan="2">performance</td><td>approximately</td><td>a</td><td> 10%,</td>

<td>approximately</td><td>a</td><td> 20%,</td><td>approximately</td><td>a</td><td> 30%,</td>

<td>approximately</td><td>a</td><td> 40%,</td><td>approximately</td><td>a</td><td> 50%,</td>

<td>approximately</td><td>a</td><td> 60%,</td><td>approximately</td><td>a</td><td> 70%,</td>

<td>approximately</td><td>a</td><td> 80%,</td><td>approximately</td><td>a</td><td> 90%,</td>

approximately 100%, approximately 200%, approximately 300%, approximately 400%, approximately 500% or more, compared to carrying out the reaction essentially under the same conditions but in the absence of a salt.

As one skilled in the art will understand, during fluoridation, any associated anionic species can be exchanged (e.g., in cases where the starting material is a salt). That is, the starting material can be provided as a first salt (e.g., trifluoroacetate, chloride) and the isolated product (e.g., the fluorinated product) can be isolated as a second different salt (e.g. ., formate, ascorbate, citrate or trifluoroacetate). In some cases, after the formation of a salt, the counter-anion can be exchanged in an additional reaction step. For example, the hydrochloric salt of a compound can be exposed to a suitable reagent (e.g., AgOAc or AgOBz) so that the compound forms the corresponding reagent salt (e.g., acetate salt or benzoate salt , respectively). As another example, the trifluoroacetic salt of a compound can be exposed to a suitable reagent (e.g. , phosphoric acid or methanesulfonic acid) so that the compound forms the corresponding reagent salt (e.g., phosphate salt or methanesulfonate salt, respectively). The intermediate salt (e.g., trifluoroacetate chloride salt in the previous examples) may or may not be isolated before being exposed to the reagent.

Those skilled in the art will be able to select and / or determine the appropriate set of reaction conditions (e.g., concentration, temperature, pressure, reaction time, solvents) that are suitable for use in a particular application. The tracing agent can be further processed using one or more purification techniques, and can optionally be combined with additional components such as a stabilizing agent.

In some embodiments, the tracing agent is formed as a salt (eg, a pharmaceutically acceptable salt).

In some embodiments, a formate salt comprising formula (VI) is provided:

<img file="MX367382B_D0077.tif" />

where X® is formate.

In other embodiments, an ascorbate salt is provided comprising the formula (VII)_:

<img file="MX367382B_D0078.tif" />

where X® is ascorbate.

In other embodiments, a citrate salt is provided comprising the formula:

<img file="MX367382B_D0079.tif" />

where Χθ is citrate.

In other embodiments, a trifluoroacetate salt comprising the formula is provided:

<img file="MX367382B_D0080.tif" />

where X® is trifluoroacetate.

In certain embodiments, the fluorine in the salt of formula (I), (VI), (VII), (ix) or (X) is isotopically enriched with F. In some embodiments, a pharmaceutically acceptable composition is provided.

In certain embodiments, the pharmaceutically acceptable composition comprises a salt comprising the formula (VI):

<img file="MX367382B_D0081.tif" />

where X® is formate or a salt comprising the formula (VII):

where X ®

<img file="MX367382B_D0082.tif" />

It is ascorbate combinations of these, and optionally a pharmaceutically acceptable excipient. Other pharmaceutically acceptable compositions comprise the citrate salt of formula (IX) or the trifluoroacetate salt of formula (X)_#.

Pharmaceutically acceptable excipients and other aspects of the pharmaceutically acceptable compositions are described herein.

It was found that the formate salt and the ascorbate salt had unexpected properties, including improved purity and / or stability compared to other salt forms of the compound comprising the formula (VIII):

Br ·

NH₂® θχ

NH₂

H ²

0 '(VIII), where X® is a counter-anion.

In addition, in some cases, it has been found that the salt form of the precursor of the compound of formula (VI) or (VII) can affect the purity of the final product in a pharmaceutically acceptable composition (e.g., for use as a tracing agent ). For example, with respect to the formate salt (i.e., a compound of formula (VI)), it has been found that this salt form has unexpected characteristics with respect to purification (e.g. eg, the compound can be isolated more easily and / or with higher yields compared to other salt forms). This may be due to the solubility characteristics of the salt. In addition, it has been found that the salt form has unexpected characteristics with respect to stability. In some embodiments, ascorbate salts of isotopically enriched trace agents in¹⁸F are substantially more stable compared to other salt forms.

In some embodiments, the conversion of a compound of formula (VIII) into a compound suitable for use in a pharmaceutically acceptable composition involves three steps: (1) purification (e.g., by HPLC), (2) solvent exchange and (3) formulation. In some cases, the compound of formula (VIII) is purified by HPLC, and the purification, retention and / or resolution of the compound is sensitive to the pH and / or the buffering capacity of the mobile phase. There may be several reagents contained in the mobile phase to purify the compound efficiently, including acetic, citric and / or formic acid modifiers. In a particular embodiment, the presence of formic acid in the mobile phase is particularly effective. In addition, it was also discovered that the additive affects solvent exchange, since elution of a compound (eg, through a C18 Sep-Pak) may depend on the composition of the mobile phase. In some cases, the salt formulation may be affected by both the pH of the solution and the identity of the salt form. The pH can be adjusted to control acute radiolytic decomposition during solvent exchange, while counter-anion selection can be based on long-term antioxidant capacity.

Those skilled in the art will be able to select a source of a fluoride species that is suitable for use in the methods described herein. The term "fluoride species", as used herein, refers to a fluoride atom or a group of atoms comprising at least one fluoride atom, where the fluoride atom is capable of reacting with another compound (e.g. eg, a precursor of the tracer agent). In some modalities, a kind of ^ieF isotopically enriched by nuclear reaction ¹⁸O (p, n)¹⁸F from proton bombardment of [¹⁸O] H2O in a cyclotron. The method may involve treating a solution of the species of¹⁸F to remove impurities, such as [¹⁸O] H2O that has not reacted. For example, you can filter a solution of the kind of¹⁸F through an anion exchange column, where the species of ^1SF is retained in the cationic resin matrix while the [¹⁸O] H2O elutes. Then the kind of¹⁸F is removed by washing the anion exchange column with various mixtures of solvents and optional reagents (eg, salts), to form a solution containing ¹⁸F. In some cases, the column of

<img file="MX367382B_D0083.tif" />

Anion exchange is washed with an aqueous solution of a salt such as K2CO3 or Et₄NHCO3. In other cases, the column is washed (e.g., with K₂CO₃ aqueous), and the resulting solution is diluted (e.g., with MeCN) and / or concentrated (e.g., to dryness using elevated temperature and / or reduced pressure). You can get [¹⁸F] KF and / or [¹⁸F] Et₄Anhydrous NF and can be reacted with a compound or a salt thereof.

In some cases, the solution it contains ¹⁸F is combined with additional components before reacting with a tracer agent precursor. For example, one or more solvents can be added to dilute the solution containing^ieF to a desired concentration. In certain modalities, the solution it contains¹⁸P is diluted with acetonitrile (MeCN) In certain embodiments, the solution containing ¹⁸F is diluted with acetonitrile (MeCN) and t-BuOH.

In some cases, the solution it contains ¹⁸F can be concentrated to dryness by exposing it to an elevated temperature and / or reduced pressure to form an anhydrous solid containing F. In some embodiments, the solid containing 18

F may further comprise one or more reagents (eg, salts). The chemical composition of the solid it contains^youF may depend on the number and type of reagents used in the preparation of the solution it contains. ¹⁸F. For example, a solution of potassium carbonate can be used to elute the species of ¹⁸F of the anion exchange column, which would result in a solid containing ¹⁸F and that comprises [¹⁸F] KF. In another example, a tetraethylammonium bicarbonate solution is used to elute the species of¹⁸F of the anion exchange column, whereby a solid containing ¹⁸F and that comprises [¹⁸F] Et₄NF

In some cases, the solution comprising the kind of ¹⁸F is heated to a temperature between room temperature and about 200 'C. For example, a solution comprising fluoride [^youF] up to high temperatures to favor evaporation of the solvent (eg, up to about 110 ° C). In some embodiments, the solution is heated to a temperature between about 90 and 120 "C or between about 100 and 150 'C. In some cases, the solution is heated to about 75 'C, about 85 ° C, about 95' C, about 105 'C,

<td>approximately</td><td> 115</td><td>'C,</td><td colspan="3">approximately 125 'C or</td><td>plus .</td><td>In</td>

<td>some cases,</td><td colspan="3">the solution is</td><td colspan="2">put under pressure</td><td colspan="2">reduced</td>

<td colspan="2">approximately</td><td> 100</td><td>mm Hg,</td><td>approximately</td><td> 125</td><td>mm</td><td>Hg,</td>

<td>approximately</td><td> 150</td><td>mm</td><td>Hg,</td><td>approximately</td><td> 175</td><td>mm</td><td>Hg,</td>

<td>approximately</td><td> 200</td><td>mm</td><td>Hg,</td><td>approximately</td><td> 225</td><td>mm</td><td>Hg,</td>

<td>approximately</td><td> 250</td><td>mm</td><td>Hg,</td><td>approximately</td><td> 275</td><td>mm</td><td>Hg,</td>

<td>approximately</td><td> 300</td><td>mm</td><td>Hg,</td><td>approximately</td><td> 325</td><td>mm</td><td>Hg,</td>

100 approximately 350 mm Hg, approximately 375 mm Hg, approximately 400 mm Hg or greater. In some cases, the solution is subjected to a reduced pressure of approximately 10,000, approximately 125mbars, approximately ISOmbarres, approximately 175 tons, approximately 200

<td>mbars,</td><td>approximately</td><td> 225</td><td>mbars,</td><td>approximately</td><td> 250</td>

<td>mbars,</td><td>approximately</td><td> 275</td><td>mbars,</td><td>approximately</td><td> 280</td>

<td>mbars,</td><td>approximately</td><td> 300</td><td>mbars,</td><td>approximately</td><td> 325</td>

<td>mbars,</td><td>approximately</td><td> 350</td><td>mbars,</td><td>approximately</td><td> 375</td>

<td>mbars,</td><td>approximately</td><td> 400</td><td>mbars,</td><td>approximately</td><td> 450</td>

mbar, approximately 500 mbar or higher. Those skilled in the art will be able to select and / or determine the appropriate conditions for a particular process. In some embodiments, the solution is concentrated to dryness at approximately 150 mm Hg and approximately 115 'C. In some embodiments, the solution is concentrated to dryness at approximately 375 mm Hg and approximately 115 'C. In some embodiments, the solution is concentrated to dryness at about 400 mbar and about 110-150 'C. In some embodiments, the solution is concentrated to dryness at approximately 280 mbar and approximately 95-115 'C.

Next, the fluoride species and / or the reagent, where appropriate, are contacted with the tracer agent precursor under conditions that result in the conversion of the tracer agent precursor into the product

101 tracing agent by nucleophilic fluorination. Those skilled in the art will be able to select the appropriate conditions for use in a particular reaction. For example, the proportion of the fluoride species against the tracer agent precursor can be selected to be about 1:10 000 or more, about 1: 5000 or more, about 1: 3000 or more, about 1: 2000 or higher, about 1: 1000 or higher, about 1: 500 or higher, about 1: 100 or higher, about 1:50 or higher, about 1:10 or higher, about 1: 5 or higher, or, in some cases, approximately 1: 1 or higher. In some embodiments, the fluoride species may be present in a proportion of approximately 10% mol, or approximately 5% mol, or approximately 3% mol, or approximately 2% mol, or approximately 1% mol, or approximately 0.5 mol%, or approximately 0.1 mol%, or approximately 0.05 mol%, or approximately 0.01 mol% in relation to the amount of precursor of the tracer. In some embodiments, the fluoride species is isotopically enriched with ¹⁸F. For example, the proportion of the species of ¹⁸P versus the precursor of the tracing agent can be selected to be about 1: 1,000,000 or more, or about 1: 500,000 or more, or about 1: 250,000 or more, or about

102

1: 100,000 or more, or about 1:50,000 or more, or about 1:25,000 or more, or about 1:10,000 or more, about 1: 5000 or more, about 1: 3000 or more, about 1 : 2000 or higher, approximately 1: 1000 or greater, approximately 1: 500 or greater, approximately 1: 100 or greater, approximately 1:50 or greater, approximately 1:10 or greater, approximately 1: 5 or greater, or, in some cases, approximately 1: 1 or higher.

In some embodiments, the nucleophilic fluorination reaction is carried out in the presence of one or more solvents, for example, an organic solvent, a non-organic solvent (eg, an aqueous solvent) or a combination of these. In some cases, the solvent is a polar solvent or an apolar solvent. In some embodiments, the solvent is an aqueous solution such as water. The solvent comprises at least about 0.001% of water, at least about 0.01% of water, at least about 0.1% of water, at least about 1% of water, at least about 5%, at least about 10%, at least about 20% water, at least about 30% water, at least about 40% water, at least about 5% water or more. In some cases, the solvent may comprise between about 0.1% and

103 approximately 100% water, between approximately 1% and approximately 90%, between approximately 1% and approximately 70%, between approximately 1% and approximately 50% or between approximately 10% and approximately 50% . In some cases, the solvent does not comprise more than about 10% water, about 5% water, about 4% water, about 3% water, about 2% water, about 1% water. water or about 0.5% water. In some cases, the solvent comprises between about 0.01% water and about 5% water, or between about 0.01% water and about 2% water, or between about 0.1% water and about 0.2 % of water.

Other non-limiting examples of solvents useful in the methods include, but are not limited to, non-halogenated hydrocarbon solvents (e.g., pentane, hexane, heptane, cyclohexane), halogenated hydrocarbon solvents (e.g., dichloromethane, chloroform, fluorobenzene, trifluoromethylbenzene), aromatic hydrocarbon solvents (e.g., toluene, benzene, xylene), ester type solvents (e.g., ethyl acetate), ether type solvents (e.g. eg, tetrahydrofuran, dioxane, diethyl ether, dimethoxyethane) and alcohol type solvents (eg, ethanol, methanol, propanol, isopropanol and

104 tert-butanol). Other non-limiting examples of solvents include acetone, acetic acid, formic acid, dimethyl sulfoxide, dimethylformamide, acetonitrile, p-cresol, glycol, petroleum ether, carbon tetrachloride, hexamethylphosphoric triamide, triethylamine, picoline and pyridine. In some embodiments, the reaction is carried out in a polar solvent such as acetonitrile. In some cases, the solvent can be selected to reduce and / or minimize the formation of secondary products. In certain embodiments, the fluorination reaction is carried out in MeCN as solvent. In certain embodiments, the fluorination reaction is carried out in t-BuOH as solvent. In certain embodiments, the fluorination reaction is carried out in a mixture of MeCN and t-BuOH as solvent. In certain embodiments, the fluorination reaction is carried out in DMF as a solvent. In certain embodiments, the fluorination reaction is carried out in DMSO as solvent. In certain embodiments, the fluorination reaction is carried out in THF as solvent.

In certain embodiments, an anhydrous solid containing may be contacted. ^ieF, which optionally comprises a reagent, with a solution of a tracer agent precursor (eg, a tosylate type precursor) and the resulting solution is heated to an elevated temperature for a selected period of time. The solution can

105 be, for example, a solution of acetonitrile. In other modalities, a solution of the species of^{1 B}F and reagent, where appropriate, with a solid tracer agent precursor or a tracer agent precursor solution.

Some embodiments involve contacting the tracer agent precursor with the fluoride species in a solution with a pH less than about 13, less than about 12 or less than about 11. In some cases, the solution has a pH between about 8 and about 9, or between about 8 and about 10 or between about 7 and about 8. In certain embodiments, the pH range for the fluorination reaction is greater than about 6, - or greater than about 7; or is between 7 and 13, inclusive; between 6 and 12, inclusive, · between 7 and 12, inclusive; between 8 and 12, inclusive; between 9 and 12, inclusive; and between 10 and 12, inclusive.

In some cases, the solution comprising the F 'species ^and^ · Precursor of the tracing agent and, optionally, a reagent, is heated to an elevated temperature for a period of time. For example, the solution can be heated to about 50 ° C, about 60 ° C, about 70 ° C, about 80 ° C, about

106

<td>90 ° C, approximately</td><td> 100</td><td>'C, approximately 110</td><td>'C,</td>

<td>about 120</td><td>'C,</td><td>approximately 150</td><td>'C,</td>

<td>approximately 170</td><td>'C,</td><td>approximately 200</td><td>'C,</td>

about 225 'C, about 250 ° C or more, for S a period of less than or equal to about 5 minutes, less than or equal to about 10 minutes, less than or equal to about 20 minutes, less than or equal to about 30 minutes. It will be understood that other temperatures and other reaction times can be used. After completion of the reaction, the reaction mixture is cooled (e.g. , to room temperature) and optionally diluted with a solvent, such as water, or solvent mixtures such as water / acetonitrile. In some embodiments, the reaction mixture is heated to elevated temperatures to favor evaporation of the solvent (eg, up to about 95 ° C). In some embodiments, the solution is heated to a temperature between about 55 and 125 'C. In some cases, the solution is heated to about 65 ° C, about 75 ° C, about 85 'C, about 95' C, about

105 'C, approximately 115 ”C or more. In some cases, the solution is subjected to a reduced pressure of approximately 100 mm Hg, approximately 12 5 mm Hg, approximately 150 mm Hg, approximately 175 mm Hg, approximately 200 mm²⁵ Hg, approximately 225 mm Hg, approximately 250 mm Hg,

107

<td>approximately</td><td> 275</td><td>mm Hg,</td><td>approximately</td><td> 300</td><td>mm Hg,</td>

<td>approximately</td><td> 325</td><td>mm Hg,</td><td>approximately</td><td> 350</td><td>mm Hg,</td>

<td>approximately</td><td> 375</td><td>mm Hg,</td><td>approximately</td><td colspan="2">4 00 mm Hg or</td>

<td colspan="2">higher. In some</td><td>cases,</td><td>the solution is</td><td>submit</td><td>to one</td>

<td colspan="2">reduced pressure</td><td colspan="2">approximately</td><td> 100</td><td>mbars,</td>

<td>approximately</td><td> 125</td><td>steals,</td><td>approximately</td><td> 150</td><td>mbars,</td>

<td>approximately</td><td> 175</td><td>nabares,</td><td>approximately</td><td> 200</td><td>mbars,</td>

<td>approximately</td><td> 225</td><td>mbars,</td><td>approximately</td><td> 250</td><td>mbars,</td>

<td>approximately</td><td> 275</td><td>mbars,</td><td>approximately</td><td> 280</td><td>mbars,</td>

<td>approximately</td><td> 300</td><td>mbars,</td><td>approximately</td><td> 325</td><td>mbars,</td>

<td>approximately</td><td> 350</td><td>mbars,</td><td>approximately</td><td> 375</td><td>mbars,</td>

<td>approximately</td><td> 400</td><td>mbars,</td><td>approximately</td><td> 450</td><td>mbars,</td>

approximately 500 robares or higher. Those skilled in the art will be able to select and / or determine the appropriate conditions for a particular process. In some embodiments, the solution is concentrated to dryness with an inert gas flow at approximately 95 ° C.

Upon completion of the fluorination reaction, the resulting tracing agent is optionally subjected to one or more purification steps. In some cases, the tracing agent can be reconstituted in a solvent before purification (eg, by chromatography such as HPLC). In some cases, the tracer agent is dissolved in water, acetonitrile or combinations thereof. In some modalities, after forming a solution comprising the tracing agent and the

108 solvent, and before purification (eg, by HPLC), the solution is heated. In a particular embodiment, the tracing agent is reconstructed in a water / acetonitrile mixture and heated (e.g., to a temperature between about 90 and 100 ° C) for about 1 minute, about 3 minutes, about 5 minutes , about 10 minutes, about 20 minutes, about 30 minutes or more. After heating the mixture, the solution can be optionally cooled before purification.

Check out

Those skilled in the art will be familiar with the appropriate conditions to deprive guanidine functional groups. As indicated below, protective groups can be removed before or after fluoridation. In some embodiments, suitable conditions comprise exposing a compound comprising a protected guanidine functional group to an acid. The acid can be added pure or in solution (e.g. eg, so that the acid has a concentration of about 0.1 M, about 0.2 M, about 0.3 M, about 0.4 M, about 0.5 M, about 0.75 M or about 1.0 M). In certain embodiments, the nitrogen protecting group is t-butyloxycarbonyl and the acid used for the deprotection step is acidic.

109 trifluoroacetic In certain embodiments, after deprotection, the compound is a salt (eg, a trifluoroacetate salt).

In some cases, suitable conditions for deprotection comprise acidic conditions. The acid can be provided in a ratio of about 2: 1, about 1: 1, about 1: 2, about 1: 3 or about 1: 4 of compound: acid. In certain embodiments, the pH range for deprotecting the precursors of tracer agents such as compounds of formula (II) (or alternatively protected fluorinated tracer agents of the invention) may be less than or equal to about 4, including the minor or equal range. to about 3, less than or equal to about 2 and less than or equal to about 1.

The conditions may comprise one or more solvents. Non-limiting examples of solvents are provided herein. The reaction can be carried out at any suitable temperature and, in certain embodiments, the deprotection reaction is carried out at a temperature greater than or equal to room temperature. The product can be analyzed, isolated and / or purified using techniques with which those skilled in the art will be familiar (e.g. eg, column chromatography, HPLC, NMR, MS, IR, UV / Vis). In some cases, the product is isolated as a salt (e.g.,

110 by filtration, crystallization). In certain embodiments, the salt is an ascorbate salt. In certain embodiments, the salt is a formate salt. In other embodiments, the salt is a citrate salt or a trifluoroacetate salt.

Purification and formulation

In some cases, the synthesis, purification and / or formulation of a tracing agent (e.g., a compound comprising formula (I) or (V)) is carried out using an automated reaction system that optionally comprises a cassette, where the cassette comprises a synthesis module, and / or a purification module, and / or a formulation module. Automated cassettes and reaction systems are described herein.

Purification and isolation can be carried out using methods known to those skilled in the art, including separation techniques, such as chromatography, or combinations of various separation techniques known in the art, for example, extractions, distillation and crystallization. . In one embodiment, high performance liquid chromatography (HPLC) with a solvent or a mixture of solvents is used as eluent to recover the product. In some cases, the eluent includes a mixture of water and acetonitrile such as a 20:80 mixture of water: acetonitrile. The water content of the eluent may be comprised,

111 for example, between about 1% and about 30%. In some cases, HPLC purification can be performed using a C18 column. The product can be analyzed (e.g., by HPLC) to determine the performance (e.g., radiochemical performance) and / or radiochemical purity.

Radiochemical purity may be greater than

<td>approximately</td><td>the</td><td> 50%,</td><td>approximately</td><td>the</td><td> 60%,</td>

<td>approximately</td><td>the</td><td> 70%,</td><td>approximately</td><td>the</td><td> 80%,</td>

<td>approximately</td><td>the</td><td> 90%,</td><td>approximately</td><td>the</td><td> 95%,</td>

<td>approximately</td><td>the</td><td> 97%,</td><td>approximately</td><td>the</td><td> 98%,</td>

<td>approximately</td><td>the</td><td>99% or</td><td colspan="2">higher. The percentage</td><td>from</td>

Product performance may be greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, approximately 60%, greater than approximately 70%, greater than approximately 75 %, greater than approximately 80%, greater than approximately 85%, greater than approximately 90%, greater than approximately 92%, greater than approximately 95%, greater than approximately 96%, greater than approximately 97% , greater than approximately 98%, greater than approximately 99% or greater. In some modalities, the radiochemical performance is between 15 and 50%.

The product can be further processed using additional purification techniques such as the

112 filtration. In some cases, the tracer agent is purified using HPLC, to produce a solution of the mobile phase of HPLC and the tracer agent. The mobile HPLC phase can then be exchanged for a solution of ascorbic acid or a salt thereof and an ethanol solution by filtration through a C-18 resin (e.g., a C18 Sep-Pak cartridge). In some embodiments, the solution of the mobile phase of HPLC and the tracing agent is filtered through a C-18 resin, so that the tracing agent remains in the resin and the other components, such as acetonitrile and / or other solvents or components, are removed by elution. The C-18 resin can be washed further with a solution of ascorbic acid or a salt thereof and the filtrate can be discarded. To recover the purified tracing agent, the C-18 resin is washed with a solvent, such as ethanol, and the resulting solution is further diluted optionally with a solution of ascorbic acid or a salt thereof, as described herein. Present.

Optionally, the recovered product is combined with one or more stabilizing agents such as ascorbic acid or a salt thereof. For example, a solution comprising the purified tracing agent can be further diluted with a solution of ascorbic acid or a salt thereof. As described herein, a formulation can be prepared using an automated reaction system comprising

113 a cassette

In some cases, a solution comprising the tracer agent product can be filtered under aseptic conditions (eg, using a Millex PVDF 0.22 pm Millipore sterilizing filter with a diameter of 13 mm) in a sterile product vial. The sterile product vial can be a previously sterilized unit that can be purchased from commercial suppliers which does not open during the production process, so that the tracing agents (or other components) can be inserted under aseptic conditions through the septum Before its use. Those skilled in the art will be able to select suitable vials and production components, including previously sterilized units that can be purchased from commercial suppliers which comprise a purge filter with a membrane with a pore size of 0.22 pm and syringes Sampling for quality control.

After aseptic filtration, individual doses can be introduced into syringes, labeled and sent to the clinical center. Dosage techniques, kits, cassettes, methods and systems (eg, automated reaction systems) for synthesizing the tracing agent and analysis procedures are described herein. In some embodiments, the product is dispensed in a 3 or 5 mL syringe and labeled for distribution.

The labels can be

114 Prepare in a radiopharmacy and can be applied to a syringe protector or a shipping container. Additional labels may be provided on the shipping container to be included in the clinical center record.

Uses of tracing agents

In another aspect, the present invention provides methods for performing a tomography, including methods for performing a tomography on a subject which include administering a composition or formulation that includes a tracing agent of the invention (i.e., a compound of formula (I ), including a compound of formula (V), such as, without limitation, tracer agent-1) to the subject by injection, infusion or any other method of administration, and perform a tomography of a region of interest of the subject. Regions of interest may include, without limitation, the heart, a portion of the heart, the cardiovascular system, the cardiac vessels, the blood vessels (e.g., arteries and / or veins), the brain, the pancreas , the adrenal glands, other organs and tumors. As described herein, the tracer-1 agent comprises the formula:

18_F

NH

<img file="MX367382B_D0084.tif" />

115 or a salt, free base or a pharmaceutically acceptable combination thereof. In some embodiments, a pharmaceutically acceptable salt of the tracer-1 agent comprises the formula:

NH₂® ®X

Y and N nh₂z where Χθ is a counter-anion. In certain embodiments, X® is formate or ascorbate. In some embodiments, X® is citrate or trifluoroacetate.

In some embodiments, the methods of this description include (a) administering to a subject a composition that includes a tracer agent of the invention including, but not limited to, tracer agent-1, and (b) acquiring at least one image of minus a portion of the subject. In some cases, the acquisition step uses positron emission tomography (PET) to visualize the distribution of the tracer agent in at least a portion of the subject. As those skilled in the art will understand, a tomography using the methods of this description may include a whole body tomography of a subject, or a tomography of a specific body region, organ or tissue of the subject of interest. For example, if it is known or suspected that a subject suffers from myocardial ischemia, the methods of this description can be used to perform a

116 tomography of the subject's heart. In some modalities, the tomography may be limited to the heart or may include the heart and vasculature associated with it.

In some embodiments, the tracing agents of the invention are used, including, but not limited to, the tracer-1 agent, to monitor and / or evaluate certain aspects of the sympathetic nervous system (SNS). SNS plays a role in normal cardiac regulation and / or the pathogenesis of development and / or the progression of heart failure. In general, after a heart injury (p. eg, myocardial infarction, valvular regurgitation, hypertension), compensatory activation of the SNS is induced to help maintain sufficient cardiac output. The increase in sustained activity of the cardiac SNS can cause an increase in the release of cardiac norepinephrine (NE), a reduction in the betal adrenergic receptor and / or a reduction in the NE transporter (NET), which may cause the infiltration of NE. High levels of NE can be attributed to cardiac myocytic hypertrophy, fibroblastic activation, collagen deposition and / or myocytic apoptosis, which can cause ventricular remodeling and / or susceptibility to arrhythmia.

In some embodiments, the evaluation of changes and / or the presence of a neurotransmitter in a subject, and certain parameters related to the neurotransmitter provide

117 information related to cardiac events. For example, the evaluation of NET in a subject can be used to provide information regarding cardiac events and / or cardiac exposure to NE. In some cases, the neurotransmitter is a monoamine other than NE.

In some embodiments, the neutrotransmitter is NE. The use of a tracer agent that has NET as a target allows images of the location, concentration, density and / or distribution of NET to be captured and can also be used to detect changes in NET over time, for example, by acquiring a first NET image in a subject or a region of a subject, · obtaining a subsequent NET image of the subject or region of the subject and comparing the first image with subsequent images. Differences between images may provide information about the change in the NET status of the subject or the region of the subject. Changes in a NET parameter (e.g., location, density, concentration and / or distribution) over time can be evaluated and correlated with the occurrence of the disease, its progression and / or its regression. In some embodiments, a method comprises administering a dose of a pharmaceutically acceptable composition (eg. , the tracer agent-1) to a subject and acquire at least one image of a portion of the subject, where the image allows to evaluate and / or detect NET in the subject. In some cases, detection includes detecting

118 the level (eg, concentration) of NET, detect the density of NET, detect the function of NET and / or detect the location of NET.

In some modalities, changes in NET (eg, density, location, concentration, function) can be used to assess the presence and / or absence of a condition, disease and / or disorder. For example, in some cases, changes in NET can be used to assess cardiac sympathetic innervation and / or myocardial sympathetic function in a subject. For example, an increase or decrease in the concentration of NET in a portion of the subject (e.g. , heart) may indicate sympathetic cardiac innervation in that portion of the subject. In some cases, subjects with altered NET functions are correlated with heart failure and / or rapid myocardial reorganization.

In some embodiments, a tracer agent that has NET as a target can also be used to observe, estimate and / or quantify localized blood flow to the tissue. More specifically, there may be cases in which the level of tracer agent (or radioactivity) observed in the myocardium has been reduced compared to normal or may be below the threshold. This signal reduction may be due to several reasons, one of which may be reduced blood flow to or through the myocardium. For

119 To determine the reason, a tomography of the subject can be performed using a different tracer agent and / or a different tomographic modality suitable for detecting blood flow. The comparison of images obtained using the different methods can reveal whether the reduction or absence of signal by the tracer agent having NET as the target is attributable to blood flow instead of a difference in level, activity or a similar parameter of NET. In other embodiments of the invention, the myocardium can be serially scanned, for example, immediately after administering the tracer agent, to observe the movement of the tracer agent into the heart. Such serial images should provide information on blood flow through the heart. Later images are also acquired as these reveal a more stable state of blood flow to the inside and outside of the heart, as well as the retention of blood in the heart. In this way, alterations in global, local or regional blood flow can be distinguished from local or regional changes in NET density, location, concentration and function described above. In some embodiments, a tracer agent having NET as a target is used to assess the ability of a therapeutic agent and / or treatment to modify NET. For example, the images acquired for

120 a subject to which a tracer agent of the invention has been administered, including, but not limited to, tracer agent-1, before therapeutic treatment can be compared with the images acquired for the same subject after therapeutic treatment for the purpose of determine if the treatment has affected the location, concentration and / or density of NET in the subject. Similarly, images obtained at different times and / or before and after treatment can be used to detect changes in NET in a subject over time and / or with treatment.

In some aspects, global images (e.g., global NET images) are acquired and, in other aspects of the invention, regional images (e.g., regional NET images) are acquired after administering a tracing agent that have NET as the target, where a global image is an image of all or virtually the entire organ (eg, heart, liver, pancreas) and a regional image is an image of only a portion of an organ. Images can be acquired using an image capture system such as a PET system, a SPECT system or any other suitable tomographic system.

In some modalities, the images can be acquired during a single time interval and, in other modalities, they can be acquired as a series of images with identical or different acquisition periods that begin

121 at the time of administration or at a later time.

In some modalities, methods are provided for diagnosis or that facilitate the diagnosis of a disease or condition, methods for assessing the efficacy of the treatment of a disease or condition, or methods for performing a tomography on a subject that is known or suspected who suffers from a cardiovascular disease or condition that alters sympathetic innervations. A cardiovascular disease can be any disease of the heart or other organ or tissue irrigated by the vascular system. The vascular system includes the coronary arteries and all peripheral arteries that supply the peripheral vascular system and the brain, as well as the veins, arterioles, venules and capillaries. In some cases, cardiac innervation can be examined, since abnormalities in cardiac innervation have been linked to the pathophysiology of many heart diseases, including sudden cardiac death, congestive heart failure, diabetic autonomic neuropathy, myocardial ischemia, and cardiac arrhythmias. Other non-limiting examples of cardiovascular diseases of the heart include diseases such as coronary artery disease, myocardial infarction, myocardial ischemia, angina pectoris, congestive heart failure, cardiomyopathy (congenital or acquired), arrhythmia or cardiac valvulopathy. In some modalities, the methods

122 described herein are useful for monitoring and measuring cardiac innervation. For example, a method described herein can determine the presence or absence of cardiac innervation. Heart conditions may include damage that has not been caused by a disease, but is the result of injury, e.g. ex. , traumatic injuries or surgical injuries. The methods described herein can be used in some modalities to determine global or regional changes in cardiac sympathetic innervation.

In some cases, a subject to whom a tracer agent of the invention can be administered may have signs or symptoms suggestive of a disease or condition associated with abnormalities in cardiac innervation. In some cases, the use of the tracing agent can be used to diagnose incipient or pre-illness conditions that indicate that a subject has a higher risk of suffering from a disease. The tomographic methods described herein can be used to detect cardiac innervation in subjects who have already been diagnosed with a disease or condition associated with cardiac innervation abnormalities, or in subjects who have no history of a disease or condition of this. type or those diagnosed with a disease or condition of this type. In other cases, the methods can be used to obtain

123 measures that provide a diagnosis or that facilitate obtaining a diagnosis of a disease or condition associated with cardiac innervation abnormalities. In some cases, the subject may already be undergoing pharmacotherapy for a disease or condition associated with cardiac innervation abnormalities, while in other cases, the subject may not be currently undergoing therapy for a disease or condition associated with abnormalities in cardiac innervation. In some embodiments, the method can be used to evaluate the efficacy of a treatment for a disease or condition. For example, the heart can be visualized using the tracing / contrast agents described herein before, during and / or after the treatment of a condition that affects the heart of a subject. This visualization can be used to evaluate a disease or condition, and can facilitate the selection of a treatment regimen, e.g. eg, therapy, surgery, medications, for the subject.

In some embodiments, a compound of the present invention is used to determine the presence or absence of a tumor in a subject. In some modalities, the tumor is a tumor that expresses NET. In some embodiments, a tracer agent of the invention is used to determine a tumor's response to therapy in a subject. The methods to determine the presence of a tumor and / or to determine the

124 A tumor's response to therapy in a subject can follow the same methods or methods similar to those described for tomographic methods in a subject.

In some embodiments, the tracer agent of the invention (e.g., tracer agent-1) is used as a tracer agent combined with positron emission tomography (PET) or with other tomographic methods, including, but not limited to, single photon emission computed tomography (SPECT). In some cases, PET tomography can be used to obtain cardiac sympathetic neuronal images in a subject after administration of the tracer-1 agent to the subject. For example, tracer-1 agent can be administered to a subject and tomography can be performed on the subject using PET. As those skilled in the art know, PET is a non-invasive technique that allows obtaining images and measurements in series in a single subject for a period of time. The PET scan used can be performed using known systems, methods and / or devices. In some embodiments, PET tomography is performed using a cardiac tomography system. A cardiac tomography system may include a PET type tomographic functionality and a control unit configured to make the tomographic functionality perform a PET type tomographic procedure.

125 in a portion of the subject of interest before, during and / or after administering the tracer-1 agent to the subject. In some cases, the control unit is configured to make the tomographic functionality perform a PET type tomographic procedure. The control unit may comprise a computer system and / or software. In this case, the computer system can be programmed or configured to execute the necessary methods to acquire and / or analyze the images. In addition, the system may include a data storage device that can be read by a machine, which encompasses a set of instructions executable by the machine to carry out the necessary methods of acquisition and / or analysis of the images.

Those skilled in the art will be familiar with tomographic systems (eg, cardiac tomographic systems) and their components. Many tomographic systems and their components are available and marketed (eg cameras, software for analyzing images), for example, the Siemens Biograph-64 scanner or other scanners suitable for tomography. In some embodiments, data on the images is acquired in list mode and such list data can be used to create static, dynamic or synchronized images. A person skilled in the art will be able to determine a suitable period of time to acquire images, and this

126 It may vary depending on the cardiac system in which the tomography is to be performed, the tracer agent (eg, amount administered, composition of the tracer agent, subject parameters, area of ​​interest). A period to acquire images or an image acquisition period, as used herein, may refer to a period of time to obtain a single continuous image and / or may refer to a period during which one or more More individual discrete images. Therefore, an image acquisition period may be a period during which one or more images of one or more regions of a subject are acquired.

The expression list mode, as used herein, is given its usual meaning in the 15. technique. With respect to PET, list mode is a way in which the data used to create a PET image can be initially collected. In list mode, a portion of matching events (that is, each portion of pairs of detected photons) or each of them generates an entry in a list of events. Each entry includes information of various types including, but not limited to, which detectors participated, the energy of the detected photons, the detection time and / or if there was a cardiac synchronization mark. The information can be converted into one or more images by the process of

127 reformatting and / or histogram construction, where a portion of the events or all of them for each pair of detectors is added, followed by the resulting set of projections (eg, in the form of a synogram in which for each cut, each horizontal line in the synogram represents the projections for matches at a given angle). The list mode can be contrasted with the histogram mode in which the sums are made during the acquisition so that the only unprocessed data is the synogram. In some modes, the histogram mode can be used.

In some embodiments, the period of image acquisition after administration of the tracer agent1 to a subject may be between about 0 seconds and about 60 minutes, between about 1 minute and about 30 minutes, between about 5 minutes and about 20 minutes, or it can be at least about 1 minute, at least about 3 minutes, at least about 5 minutes, at least about 6 minutes, at least about 7 minutes, at least about 8 minutes, at least about 9 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 30 minutes, at least about 45 minutes, at least about 60

128 minutes, at least about 90 minutes, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours or more. In some embodiments, the image acquisition period may begin before the administration of the tracer-1 agent to a subject. For example, the image acquisition period may begin more than about 10 minutes, about 5 minutes, about 4 minutes, about 3 minutes, about 2 minutes, about 1 minute or about minutes before administration of the tracer-1 agent to subject. In some modalities, image acquisition may be continuous during the image acquisition period, or they may be acquired at intervals such as synchronized or periodic tomography.

In some embodiments, a tracer agent of the invention (eg, tracer agent-1) is provided in ethanol / ascorbic acid. In some embodiments, a tracer agent of the invention (eg, tracer agent-1) is provided as a composition comprising ethanol, ascorbic acid (eg, as sodium ascorbate) and water. In some cases, the composition comprises less than about 2.0% by weight of ethanol, less than about one by weight of ethanol less than

129

<td>approximately</td><td>a</td><td>10% in</td><td>weight</td><td>from</td><td>ethanol,</td><td>less</td><td>from</td>

<td>approximately</td><td>a</td><td>8% in</td><td>weight</td><td>from</td><td>ethanol,</td><td>less</td><td>from</td>

<td>approximately</td><td>a</td><td>6% in</td><td>weight</td><td>from</td><td>ethanol,</td><td>less</td><td>from</td>

<td>approximately</td><td>a</td><td>5% in</td><td>weight</td><td>from</td><td>ethanol,</td><td>less</td><td>from</td>

<td>approximately</td><td>a</td><td>4% in</td><td>weight</td><td>from</td><td>ethanol,</td><td>less</td><td>from</td>

<td>approximately</td><td>a ;</td><td colspan="3">5% by weight ethanol</td><td>or less</td><td>ethanol.</td><td>In</td>

<td>some cases,</td><td colspan="3">, the composition</td><td colspan="2">understands</td><td>less</td><td>from</td>

<td>approximately</td><td> 100</td><td>mg / mL,</td><td>less</td><td>from</td><td colspan="2">approximately</td><td> 75</td>

<td>mg / mL, less</td><td>from</td><td colspan="2">approximately</td><td> 60</td><td>mg / mL,</td><td>less</td><td>from</td>

<td>approximately</td><td> 50</td><td>mg / mL,</td><td>less</td><td>from</td><td colspan="2">approximately</td><td> 40</td>

<td>mg / mL, less</td><td>from</td><td colspan="2">approximately</td><td> 30</td><td>mg / mL,</td><td>less</td><td>from</td>

<td>approximately</td><td> 20</td><td>mg / mL,</td><td>less</td><td>from</td><td colspan="2">approximately</td><td> 10</td>

mg / rnL or less ascorbic acid (eg, sodium ascorbate) in water. An illustrative non-limiting formulation of the tracer-1 agent includes about 5% by weight of ethanol and about 50 mg / mL of ascorbic acid. In a particular non-limiting embodiment, a compound comprising the formula (VI) or (VII) is provided as an aqueous solution comprising less than about 5-yi by weight of ethanol and less than about 50 mg / mL of sodium ascorbate in water As those skilled in the art will understand, in the presence of ascorbic acid, at least a portion of the tracer-1 agent may be present as the ascorbate salt, such that the tracer-1 agent will have the formula:

130

<img file="MX367382B_D0085.tif" />

where X® is ascorbate.

Additional components of a composition comprising a tracer agent of the invention (eg, tracer agent-1) may be selected depending on the mode of administration to the subject. One skilled in the art will know several modes of administration that effectively administer the pharmacological agents of the invention to a desired tissue, cell, organ or body fluid. In some embodiments, the tracing agent of the invention (eg. , tracer agent-1) is administered intravenously (eg, intravenous bolus injection) using methods with which those skilled in the art will be familiar. A dose administered to a subject, as used herein, refers to an amount of the tracing agent, e.g. eg, the tracer-1 agent, which is introduced into the body of the subject.

In some embodiments, the volume of the tracer agent administered may be between 0 and about 3 mL, between about 3 mL and about 5 mL, or between about 5 mL and about 10 mL.

In some modalities, due to factors such as

131 partial retention of the tracer agent, such as tracer agent-1, in a syringe, tube, needles or other instrument used to administer the tracer agent to a subject, the amount of tracer agent, such as tracer agent-1, that there is In the syringe or other instrument, as measured or determined, prepared for administration may be greater than the amount in the dose administered to the subject. In some embodiments, the injection of the tracer agent is followed by a wash injection with normal saline in the subject, using the same tube, needle, path, etc. employees for the administration of the tracer agent.

Washing can be performed immediately after administration of the tracer-1 agent or up to about 1 min, about 2 min, about 3 min, about 5 min or more after administration. In some modes, the wash can last between 0 and 10 seconds, between 10 seconds and 25 seconds, or between 25 seconds and 60 seconds.

The volume of saline or other washing agent may be up to about 5 mL, about 6 mL, about 7 mL, about 8 mL, about 9 mL, about 10 mL, about 15 mL, about 20 mL or more. As will be understood by those skilled in the art, in the modalities in which the

132 Tracer-1 agent is administered using a syringe or other container, the actual amount of Tracer-1 agent administered to the subject may be corrected taking into account any amount of Tracer-1 agent that remains in the package. For example, the amount of radioactivity that remains in the package, the tube and the needle or the delivery instrument that transferred the tracing agent from the container to the subject can be determined after the tracer agent has been administered to the subject, and The difference between the initial amount of radioactivity and the amount remaining after administration will indicate the amount that has been supplied to the subject. In some cases, the container or injection device (e.g. eg, catheter, syringe) can be rinsed with a solution (eg, saline) after administration of the tracer-1 agent.

A composition of a tracer agent of the invention (eg, tracer agent-1) can be prepared for injection into an injection syringe. The tracing agents can be prepared in a radiopharmacy (e.g., using the methods described herein) and / or in a PET manufacturing center, and can be provided to the healthcare professional for administration. . A dose of the tracer-1 agent can be diluted with saline solution (e.g. , as described herein), if a practical dose volume is needed. For example, if the concentration

133 of activity for the tracer-1 agent is so high that only approximately 0.1 mL is needed for a suitable dose for a subject, the solution may be diluted, e.g. eg, with sterile saline, so that the syringe contains between about 0.5 mL and about 6 mL or more mL of a solution of the tracer-1 agent for administration. In some embodiments, the injection volume for the tracer-1 agent is between about 0.5 and about 5 mL, between about 1 and about 4 mL, between about 2 and about 3 mL, is at least about 0.5 mL, about 1 mL, about 2 mL ·, about 3 mL, about 4 mL, about 5 mL, about 6 mL, about 7 mL, about 8 mL ·, about 9 mL, approximately 10 mL or higher. Those skilled in the art will know how to dilute the tracer-1 agent to produce a sufficient dose volume for administration. In some aspects, the tracer-1 agent is provided in a container, such as a vial, a bottle or a syringe, and can be transferred, as appropriate, to a suitable container such as a syringe for administration.

The components of a composition comprising a tracer agent of the invention (e.g., tracer agent1) may be selected depending on the mode of

134 administration to the subject Those skilled in the art will know several routes of administration that effectively deliver the tracing agents of the invention to a desired tissue, cell, organ or body fluid. In some embodiments, the tracer agent is administered intravenously (eg, intravenous bolus injection) using methods that those skilled in the art will be familiar with.

The useful dose of tracer agent to be administered and the particular route of administration will vary depending on factors such as age, weight and the particular region in which the tomography is to be performed, as well as the particular tracer agent employed. , the contemplated diagnostic use and the form of the formulation, for example, suspension, emulsion, microsphere, liposome or the like, as described herein, and as will be apparent to those skilled in the art.

In one embodiment, the tracer-1 agent is administered by intravenous injection, usually in saline solution, at a dose between about 0.1 and about 20 mCi (and all combinations and sub-combinations of specific dose and dose ranges within these ranges, and as described below) or between a dose of about 0.5 and about 14 mCi. The tomography is performed using techniques that will be

135 A person skilled in the art and / or as described herein is familiar.

Based on dosage studies, the maximum desirable dose administered to a subject can be based on the determination of the amount of tracer agent of the invention (e.g., tracer agent-1) that limits the radiation dose to approximately 5 rem in the critical organ (e.g., urinary bladder) and / or an effective dose (ED) of about 1 rem or less, as the 10 experts in the art will understand. In some embodiments of the invention, the maximum desirable dose or the total amount of tracer-1 agent administered is between about 8 mCi and about 13 mCi. In some embodiments of the invention, the maximum desirable dose or the total amount of tracer-1 agent administered is between about 10 mCi and about 13 mCi. In some embodiments of the invention, the maximum desirable dose or the total amount of tracer-1 agent administered is between about 8 mCi and about 10 mCi. In some embodiments, a desirable dose may be less than or equal to about 15 mCi, less than or equal to about 14 mCi, less than or equal to about 13 mCi, less than or equal to about 12 mCi, less than or equal to about 11 mCi, or less than or equal to approximately 10 mCi in a period of time of

136

<td colspan="6">up to about 10 minutes, about 30 minutes,</td>

<td>approximately</td><td> 1</td><td>time,</td><td>approximately</td><td> 2</td><td>hours,</td>

<td>approximately</td><td> 6</td><td>hours,</td><td>approximately</td><td> 12</td><td>hours,</td>

approximately 24 hours or approximately 48 hours. In some embodiments, the maximum dose of the tracer-1 agent administered to a subject may be less than about pg per approximately 50 kg of body weight per day. That is, in some embodiments of the invention, the maximum dose of a composition comprising the tracer-1 agent administered to a subject may be less than about 0.28 pg of a tracer-1 agent per kg of body weight per day.

In some embodiments, the total amount of the tracer-1 agent administered to a subject is between about 0.1 mCi and about 30 mCi or between about 0.5 mCi and about 20 mCi. In some embodiments, the total amount of tracer-1 agent administered to a subject is less than or equal to about 50 mCi, less than or equal to about 40 mCi, less than or equal to about 30 mCi, less than or equal to about 20 mCi, less or equal to approximately 18 mCi, less than or equal to approximately 16 mCi, less than or equal to approximately mCi, less than or equal to approximately 14 mCi, less than or equal to approximately 13 mCi, less than or equal to approximately 12 mCi, less than or equal to approximately 10 mCi, less than or equal

137 at about 8 tnCi, less than or equal to about 6 mCi, less than or equal to about 4 mCi, less than or equal to about 2 mCi, less than or equal to about 1 mCi, or less than or equal to about 0.5 mCi. The total amount administered can be determined based on a single dose or multiple doses administered to the subject within a period of time of up to or at least about 30 seconds, about 1 minute, about 10 minutes, about 30 minutes, about 1 hour , about 2 hours, about 6 hours, about 12 hours, about 24 hours, about 48 hours or about 1 week.

In some aspects of the invention, between about 10 and about 13 mCi, or between about 8 and about 10 mCi of the tracer-1 agent are administered to a subject, and a first period of image acquisition begins at the time of administration ( p. eg, injection) or begins more than about 0 minutes, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes before administration of the tracer-1 agent. In some embodiments of the invention, the first image acquisition continues for at least about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes,

138

<td>approximately</td><td> 45</td><td>minutes</td><td>approximately</td><td> 60</td><td>minutes</td>

<td>approximately</td><td> 75</td><td>minutes</td><td>approximately</td><td> 90</td><td>minutes</td>

<td>approximately</td><td> 105</td><td>minutes</td><td>approximately</td><td> 120</td><td>minutes or</td>

plus. After the first period of image acquisition, the subject may be subjected to one or more additional image acquisition periods for up to about 1,^toabout 2, about 3, about 4, about 5, about 6 or more hours after administration of the tracer-1 agent. One or more additional image acquisition periods may have a duration between approximately 3 and

<td>approximately</td><td> 40</td><td>minutes between</td><td>approximately</td><td> 5</td><td>Y</td>

<td>approximately</td><td> 30</td><td>minutes between</td><td>approximately</td><td> 7</td><td>Y</td>

<td>approximately</td><td> 20</td><td>minutes between</td><td>approximately</td><td> 9</td><td>Y</td>

<td>approximately</td><td colspan="2">15 minutes, and it can be</td><td colspan="2">approximately</td><td> 10</td>

minutes The subject, in some embodiments, may return one, two, three or more times for additional tomography after the first injection of the tracer-1 agent, and a second, third or more injections of the tracer-1 agent may be administered. A non-limiting example of a method of administering and acquiring images for the tracer-1 agent in a subject comprises injecting between about 10 and about 13 mCi, or between about 8 and about 10 mCi of the tracer agent-1 to the subject, beginning the image acquisition

139 less than about 10 minutes before injection and continuing for about 60 minutes. In some embodiments, the * subject undergoes additional image acquisition for approximately 10 minutes, or for approximately 20 minutes, or for approximately 30 minutes, or for approximately 40 minutes, or for approximately 50 minutes, or for approximately 60

<td>minutes, about 1 hour, or about 2 hours, or</td>

<td>about 3 hours, or about 4 hours and</td>

<td>about 4 hours, or about 5 hours, or</td>

<td>about 6 hours, or about 7 hours, or</td>

<td>approximately 8 hours after injecting the agent</td>

tracer-1.

In some embodiments, studies may also be carried out using a specialized agent for tissue blood flow using methods that those skilled in the art will be familiar with. The images of these studies can then be used to distinguish anomalies observed in the images, for example, of agent-1, due to changes in NET from those due to alterations in global, regional or local blood flow.

Examples of cassettes and reaction systems

In some embodiments, systems, methods, kits and cassettes for synthesizing a tracer agent are provided.

140 the invention (eg, tracer-1 agent). In some embodiments, a tracing agent can be prepared using an automated reaction system comprising a disposable or disposable cassette. The cassette may comprise all non-radioactive reagents, solvents, tubes, valves, reaction vessels and other apparatus and / or components necessary to carry out the preparation of a particular batch of tracing agent. The cassette makes it possible for the reaction system to have versatility to prepare a variety of different tracing agents with a minimal risk of cross contamination, simply by changing the cassette. The term cassette refers to a piece of an apparatus designed to fit removably and interchangeably in automated reaction systems, so that the mechanical movement of moving parts of the automated reaction system controls the operation of the cassette from outside the cassette, that is, externally. In certain embodiments, a cassette comprises a linear arrangement of valves, each of which is connected to an inlet to which several reagents, cartridges, syringes and / or vials can be attached, either by puncturing with a needle of a vial sealed with a septum or by embedded joints that do not allow gas to escape. Each valve may have a male-female joint that interacts with a corresponding mobile arm of the

141 automated synthesizer External rotation of the arm can control the opening or closing of the valve when the cassette is attached to the automated reaction system. Other mobile parts of the automated reaction system are designed to engage the tips of the syringe plungers and thereby raise or lower the syringe cylinders. An automated reaction system may further include a controller and one or more controllable valves electrically communicated with the controller. An automated reaction system can also include vessels, valves, sensors, heaters, pressurization elements, etc. additional, electrically communicated with the controller. An automated reaction system can be operated by a controller, using software suitable for control of the opening and closing of the valves, heating, cooling, pressure levels, fluid movement, flow rate, etc. The automated reaction system may optionally include a computer operating system, software, controls, etc. or other components. In addition, the automated reaction system may comprise a support for the cassette.

Examples of automated reaction systems (e.g., a nucleophilic reaction system) include, without limitation, the Explora GN or RN synthesis system.

142 (Siemens Medical Solutions USA, Inc.), GE-Tracerlab-MX synthesis system (GE Healthcare), Eckert &amp; Zeigler ModularLab synthesis system, etc., which are normally available at PET manufacturing facilities.

Automated reaction systems can perform numerous steps, as indicated in Figure 2, which include, without limitation, the preparation of the fluoride species ^{1 B}F and a tracer agent precursor, optionally in solution (e.g., as described herein, for example, the precursor to tracer agent-1 in acetonitrile), a radiolabel reaction (e.g., the reaction of the kind of ^ieF and the tracer agent precursor to form the tracer agent), optionally in a synthesis module, purification (e.g., by preparative HPLC), solvent exchange (e.g., by SepPak), aseptic filtration and release into a container.

In some embodiments, the automated reaction system may employ a cassette comprising a reaction module fluidically connected to a purification module and / or a formulation module. Figures 3 and 4 show schematic representations of cassettes connected to illustrative reaction systems for synthesizing a tracing agent, comprising a reaction module, a purification module and / or a formulation module. Figure 5 shows a schematic representation of a system of

143 Illustrative reaction to synthesize a tracer agent comprising a reaction module. For example, the reaction module may include a reaction chamber in which the conversion of the tracer agent precursor into the tracer agent is carried out. The reaction module may include a source of a fluoride species (e.g.,¹⁸F), a source of the tracer agent precursor, a source of a reagent (e.g., a salt) and other sources of additional components such as solvents, each of which may optionally be fluidically connected to the reaction chamber . The reaction module may also comprise an anion exchange column to purify the fluoride species, before introducing it into the reaction chamber.

At the end of the reaction, the resulting tracer agent product is transferred from the reaction module to the purification module for further processing, treatment and / or purification. The purification module may include, for example, a column (eg, an HPLC column) physically connected to one or more solvent sources that are used as eluents. The purification module may further comprise a source of a stabilizing agent (e.g. eg, ascorbic acid or a salt thereof), which can be added to the tracing agent after purification (eg, by HPLC). Then, the purified tracer agent is transferred to the formulation module, where it can be carried out

144 subsequent purification and formulation. The formulation module may include a filter for aseptic filtration and / or a C-18 column for solvent exchange.

In another embodiment, a cassette comprises a reaction module and a formulation module. A reaction module of the invention may include a source of¹⁸F, an anion exchange to eliminate the (¹⁸0] H2O that has not reacted, a source of an ammonium salt, a source for a diluent for the ^iaF, a source for a tracer agent precursor (e.g., precursor to tracer agent-1 shown in Figure 1 or another precursor to tracer agent), a source for a MeCN / H2O diluent for the mixture reaction, a reaction vessel to react the ^theF and the tracer agent precursor, a solid phase extraction column (eg, a C18 column or other suitable column) fluidly communicated with the reaction vessel. The anion exchange column includes a solid sorbent to adsorb the^iaF. The [¹⁸O] H₂Or that it has not reacted and the residual reaction impurities pass through the cationic resin matrix without adsorbing on the sorbent. The reaction module also includes a source of wash solutions fluidically communicated with the anion exchange column in order to provide wash solutions to elute the^iaF of the sorbent, and includes a source

145 of an eluent (p. e j., as H₂O / MeCN or other suitable eluent comprising a salt) fluidly communicated with the anion exchange column to elute the tracer agent product from the sorbent. The reaction module may also include a source of a diluent for the¹⁸F eluted.

A formulation module of an apparatus of the invention may be fluidly communicated with a reaction module and may include a solid phase extraction cartridge that includes a solid sorbent (e.g., C-18 or other suitable sorbent) to adsorb the diluted tracer, a source of washing solutions (e.g. eg, comprising ascorbic acid, a salt of this or other suitable washing solution) fluidly communicated with the solid phase extraction cartridge in order to provide washing solutions to wash away the impurities remaining in the sorbent, and a eluent fluid source (P · ^andí · i ethanol / H₂0 or other suitable eluent fluid) fluidly communicated with the solid phase extraction cartridge to elute the tracer agent product from the sorbent. The formulation module may also include a source of a diluent (eg, comprising ascorbic acid, a salt of this or other suitable diluent) to dilute the eluted tracer. The formulation module can also be fluidly communicated with a sterilizing filter (e.g. e.g., a Millex GV PVDF sterilizing filter

146 of Millipore or other suitable sterilizing filter).

In some embodiments, a general method for synthesizing a tracer agent of the invention (eg, tracer agent-1) using an automated synthesis module is as described below. A kind of fluoride is provided [¹⁸F] (eg, in an aqueous solution) to a synthesis module. In some cases, the fluoride species (eg, in an aqueous solution) is filtered through an anion exchange column to remove the [¹⁸O] H₂Or that has not reacted, where the fluoride species [^{1 B}F] is retained in the cationic resin matrix. The column is washed with a solution (eg, an aqueous base) to elute the fluoride species [¹⁸F] inside a reaction vessel. The resulting solution is diluted (e.g., with MeCN) and then concentrated to dryness (e.g., using a high temperature and reduced pressure). The resulting material is exposed to a solution of a tracer agent precursor (eg, tracer agent precursor-1) optionally in the presence of one or more reagents (eg, an activating agent). The solution is optionally heated for a period of time (e.g. e.g., up to 90-110 ° C and kept 5-15 min) and then cooled. The solution is evaporated to dryness (e.g., using elevated temperature and / or reduced pressure), then reconstituted in a reconstitution solution (e.g.

147

H₂O / MeCN) and then purified (e.g., by HPLC on an Agilent BONUS-RP column) using a selected eluent (e.g., an NH solution₄HCO₂ in H₂0 / MeCN). The product is collected, optionally diluted (e.g., with a solution of ascorbic acid) and then transferred to a formulation module.

In a particular embodiment, a cassette is provided for use with an automated synthesis module, for example, a GE TRACERlab MX synthesis module. In one embodiment, a cassette comprises a disposable sterile assembly of distributors with molded stopcocks, specifically designed for use with the automated synthesis module (e.g., the GE TRACERlab MX synthesis module). The individual distributors are connected in a linear or non-linear manner to form a directional grouping that dictates the flow path of the reagents used in the preparation of a tracer agent (eg, tracer agent-1). In some embodiments, the cassette main body contains at least one distributor comprising a plurality of distributor positions (e.g., stopcocks). For example, the main body may comprise at least one, two, three, four or more distributors. The cassette can comprise between 1 and 20 positions of the distributor, between 1 and 15 positions of the distributor, between 5 and 20 positions of the distributor, between

148 and 15 positions of the distributor. Each of the distributors can be symmetric or not. In one embodiment, the cassette main body contains three plastic distributors, each equipped with five standard molded stopcocks, which contains 15 positions of the distributors in total. The individual stopcocks are adapted with luer accessories so that they can accommodate solvents, reagents, syringes, tubes necessary for the handling of gases and liquids, etc. The stopcocks are adapted for solvents and reagents, and can be provided with plastic tips on which inverted perforated vials are placed, while those corresponding to tubes and syringes are provided with male luer connections according to their function. In some embodiments, the cassette comprises a linear arrangement of a plurality of distributors with stopcocks connected to one or more of the components selected from the group consisting of a gas inlet, an anion exchange cartridge, a C-18 cartridge, a syringe, a solvent tank, a reaction vessel, an HPLC system, a collection vessel, a reservoir for an ascorbic acid solution or a salt thereof, and a vent outlet. In some cases, the cassette also comprises tubes. In some cases, the cassette further comprises a synthesis module of the tracer agent, where the apparatus is

149 fluidly connected to the cassette. In some cases, the apparatus is capable of carrying out the method of synthesis of a tracer agent described herein (eg, a method of synthesis of tracer agent-1).

A non-limiting example of a cassette configuration that can be used to prepare the tracer-1 agent is depicted in Figure 3. Below is a description of the connections for each of the 15 positions of the distributors: 1) connections luer gas inlet and recovery of [¹⁸O] H₂OR; 2) anion exchange cartridge - QMA Light; 3) SWFI tip connection; 4) syringe - which contains H₂O and / or MeCN; 5) luer connection

- precursor of the tracer-1 agent; 6) luer connection reaction vessel; 7) HPLC input; 8) luer connection

- ethanol; 9) luer-ascorbic acid connection; 10) luer connection - collecting vessel; 11) luer - vial connection of the final product; 12) luer connection - entrance to the column Light Sep Pak tC18; 13) luer connection - light Sep Pak tC18 column outlet; 14) syringe - containing ascorbic acid; 15) luer connections - reaction and ventilation vessel. Distributor one (pass keys 1-5) is connected to distributor two (pass keys 6-10) and distributor two is connected to distributor three (pass keys 11-15) using two male luer connections provided with a pipe Short silicone The connections of the

150 Individual distributors, luer accessories and all silicone tubes can be easily purchased from commercial suppliers.

Another non-limiting example of a cassette configuration that can be used to prepare the tracer-1 agent is depicted in Figure 4. Below is a description of the connections for each of the 15 positions of the distributors: 1) connections luer gas inlet and recovery of [¹⁸O] H₂OR; 2) anion exchange cartridge - QMA Light; 3) MeCN tip connection; 4) syringe - empty; 5) connection of the tip - precursor of the tracer-1 agent (eg, in MeCN); 6) luer connection reaction vessel; 7) HPLC input; 8) tip connection - ascorbic acid, · 9) luer connection - collecting vessel; 10) syringe - containing ethanol and / or SFWI; 11) luer - vial connection of the final product; 12) tip connection - H₂O and / or MeCN; 13) connection of the tip - ascorbic acid; 14) syringe - empty; 15) connections luer reaction and ventilation vessel. Distributor one (pass keys 1-5) is connected to distributor two (pass keys 6-10) using two male luer connections provided with a short silicone tube. Distributor two is connected to distributor three (stopcocks 11-15) using a Sep-Pak® tC-18 and suitable luer adapters. The connections of the individual distributors, the

151 Luer accessories and all silicone tubes can be easily purchased from commercial suppliers.

In some embodiments, the present invention provides a cassette for the preparation of a tracer agent comprising the formula:

NH

<img file="MX367382B_D0086.tif" />

or a salt, free base and / or pharmaceutically acceptable formula or combination thereof.

Pharmaceutical compositions

Once a compound of the present description has been prepared or obtained (e.g., a compound of formula (I), (V), (VI), (VII), (IX) or (X))), This may be combined with one or more pharmaceutically acceptable excipients to form a pharmaceutical composition that is suitable for administration to a subject, including a human being. One skilled in the art will realize that the excipients can be selected, for example, depending on the route of administration, as described below, the agent to be supplied, the duration of delivery of the agent and / or the health / condition of the subject. The pharmaceutical composition may be a solid or a liquid.

The pharmaceutical compositions of the present invention and for use in accordance with the present invention may

152 include a pharmaceutically acceptable carrier or excipient. The terms pharmaceutically acceptable excipient or pharmaceutically acceptable carrier, as used herein, refer to a filler, diluent, encapsulating or adjuvant material of solid, semi-solid or liquid, inert and non-toxic formulation of any kind. Some examples of materials that can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; tragacanth powder; malt; jelly; talcum powder; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; Sesame oil; olive oil; corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; detergents such as Tween 80; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline solution; Ringer's solution; ethyl alcohol; and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents,

153 Release agents, coating agents, sweeteners, flavoring agents and perfumes, preservatives and antioxidants may also be present in the composition, according to the formulator's criteria.

Pharmaceutically acceptable excipients include any and all of the following: solvents, diluents or other liquid carriers, dispersion or suspension adjuvants, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, which are suitable for Particularly desired pharmaceutical form. General considerations in the formulation and / or manufacture of pharmaceutical composition agents can be found, for example, in Remington's Pharmaceutical Sciences, Sixteenth Edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980) and Remington: The Science and Practice of Pharmacy, 21.^to edition (Lippincott Williams &amp; Wilkins, 2005).

The pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology. In general, such preparatory methods include the steps of associating the compound of the present invention (the active ingredient) with a carrier and / or one or more additional ingredients, and then, if necessary and / or desirable, forming and / or packaging the

154 product in a desired single- or multi-dose unit.

The pharmaceutical compositions can be prepared, packaged and / or sold in bulk, as a single unit dose and / or as a plurality of single unit doses. A unit dose, as used herein, refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active substance. The amount of active ingredient is generally equal to the dose of the active ingredient that would be administered to a subject and / or a convenient fraction of such dose such as, for example, half or a third of the dose.

The relative amounts of the active ingredient, the pharmaceutically acceptable excipient and / or any additional ingredients in a pharmaceutical composition of the invention will vary depending on the identity, size and / or condition of the treated subject and also depending on the route through the which should be administered the composition. By way of example, the composition may comprise between 0.1% and 100% (w / w) of active ingredient.

Pharmaceutically acceptable excipients used in the manufacture of the pharmaceutical compositions provided include inert diluents, dispersing and / or granulating agents, surfactants and / or emulsifiers, disintegrating agents, preservative binding agents, buffering agents, lubricating agents

155 and / or oils. In the composition, excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweeteners, flavorings and perfumes may also be present.

Illustrative diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, sodium hydrogen phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride , dried starch, corn starch, powdered sugar and combinations of these.

Illustrative conservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcoholic preservatives, acid preservatives and other preservatives.

Illustrative antioxidants include alpha-tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulphite, propionic acid, proprium gallate, sodium ascorbate, sodium bisulfite, sodium iodide, sodium metabisulphite sodium nitrite, sodium sulphite and sodium thiosulfate.

Illustrative chelating agents include ethylenediaminetetraacetic acid (EDTA) and its salts and hydrates (e.g., sodium edetate, disodium edetate, trisodium edetate,

156 disodium calcium edetate, dipotassium edetate and the like), citric acid and its salts and hydrates (e.g. citric acid monohydrate), fumaric acid and its salts and hydrates, malic acid and its salts and hydrates, phosphoric acid and its salts and hydrates, and tartaric acid and its salts and hydrates. Illustrative antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenylethyl, ethanol, phenol, phenol, phenol, alcohol phenylmercuric nitrate, propylene glycol and thimerosal.

Illustrative antifungal preservatives include butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate and sorbic acid.

Illustrative alcoholic preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate and phenylethyl alcohol.

Illustrative acid preservatives include vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid and phytic acid.

157

Other preservatives include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), burylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulphite, sodium metabisulphite potassium sulphite, potassium metabisulfite, Glydant Plus, Phenonip, methylparaben, Germall 115, Germaben II, Neolone, Kathon and Euxyl. In certain embodiments, the preservative is an antioxidant. In other embodiments, the preservative is a chelating agent.

Illustrative buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, Dgluconic acid , calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, calcium dibasic phosphate, phosphoric acid, calcium tribasic phosphate, calcium hydroxyphosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, mixtures of potassium phosphate, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate , sodium lactate, sodium dibasic phosphate, sodium monobasic phosphate, mixtures of sodium phosphate, tromethamine, hydroxide

158 magnesium, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, etc., and combinations thereof.

Liquid pharmaceutical forms for oral and parenteral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredients, liquid pharmaceutical forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing and emulsifying agents such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3butylene glycol, dimethylformamide, oils (e.g. eg, cottonseed oil, peanut, corn, germ, olive, castor and sesame), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and sorbitan fatty acid esters, and mixtures thereof. Apart from inert diluents, oral compositions may include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavorings and perfumes. In certain embodiments for parental administration, the conjugates of the invention are mixed with solubilizing agents such as Cremophor, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers and

159 combinations of these.

Injectable preparations, for example, sterile aqueous or oily injectable suspensions may be formulated according to the known technique, using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may be a sterile injectable solution, suspension or emulsion in a parenterally acceptable non-toxic diluent or solvent such as, for example, a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be used are water, Ringer's solution, uSP and an isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as solvents or suspending media. For this purpose, any insipid fixed oil, including synthetic mono- and diglycerides, can be used. In addition, fatty acids, such as oleic acid, are used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtering them through a filter that retains bacteria or incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium before use.

Suitable devices for use in the intradermal delivery of the pharmaceutical compositions described in

160 This includes devices with short needles such as those described in US Pat. UU. 4,886,499; 5,190,521; 5,328,483; 5,527,288; 4,270,537; 5,015,235;

5,141,496; and 5,417,662. Intradermal compositions may be administered with devices that limit the depth of effective penetration of a needle into the skin, such as those described in PCT publication WO 99/34850 and its functional equivalents. Jet injection devices that supply liquid vaccines to the dermis through a jet nozzle for liquids and / or through a needle that pierces the stratum corneum and produces a jet that reaches the dermis are suitable. Jet injection devices are described, for example, in US Pat. UU. 5,480,381; 5,599,302; 5,334,144;

5,993,412; 5,649,912; 5,569,189; 5,704,911; 5,383,851;

5,893,3 97; 5,466,22 0; 5,33 9,163; 5,312,33 5; 5,503,627;

5,064,413; 5,520,639; 4,596,556; 4.790,824; 4,941,880;

4,940,460; and PCT publications WO 97/37705 and WO 97/13537. Powder / particle delivery devices 20 that employ compressed gas to accelerate vaccines in the form of dust through the outer layers of the skin to the dermis are suitable. Alternatively or in addition, conventional syringes can be used in the Mantoux classic intradermal administration method.

²⁵ Although the descriptions of the compositions

161 Pharmaceuticals provided herein mainly refer to pharmaceutical compositions that are suitable for administration to humans, one skilled in the art will understand that such compositions are generally suitable for administration to all types of animals. Modification of pharmaceutical compositions suitable for administration to humans in order to transform them into compositions suitable for administration to various animals is a known process, and an expert veterinary pharmacologist may design and / or carry out the modification by performing routine experiments.

The pharmaceutical compositions of this invention can be administered to humans and / or animals parenterally (eg, by intravenous, intramuscular, subcutaneous or intraperitoneal injection). The route of administration will depend on the desired use, as recorded in the art. Ki ts

Systems, methods, kits and / or cassettes comprising a tracing agent or a precursor of the tracing agent are provided as described herein or a composition thereof and / or for the preparation of a tracing agent (e.g., the tracing agent-1). In some embodiments, kits are provided for administration of a tracer agent (eg, tracer agent-1). In some cases, the composition provided with the kit can be used for or

162 in the preparation of a tracer agent in order to detect, tomograph and / or monitor a disorder or condition. The kits of the invention may include, for example, a package comprising a tracing agent or a precursor to the tracing agent and instructions for use. The kits may comprise a sterile non-pyrogenic formulation comprising a predetermined amount of a tracing agent or a precursor of the tracing agent and optionally other components. A package that can be used together with a tracing agent (e.g., tracer agent-1), for example, to deliver and / or administer the tracer agent to a subject, can be a syringe, bottle, vial or tube . The instructions in a kit of the invention may refer to methods for synthesizing a tracer agent or a precursor of the tracer agent, methods for diluting the tracer agent or precursor of the tracer agent, methods for administering the tracer agent to a subject for the purpose of performing a diagnostic tomography or other instructions for use. A tracer or precursor of the tracer can be provided in a kit and additional preparations before use may optionally include diluting the tracer or precursor of the tracer to a concentration that can be used.

In some cases, a kit may also include one or more vials containing a diluent to prepare a

163 composition of a tracer agent (eg, tracer agent-1) to be administered to a subject (eg, a human being). A diluent vial may contain a diluent such as physiological serum or water to dilute the tracer-1 agent. For example, the tracer-1 agent can be packaged in a kit as a ready-to-inject formulation, or it may require some reconstitution or dilution by which a final composition / formulation is prepared for injection or infusion.

The instructions in a kit of the invention may also include instructions for administering the tracer agent to a subject and may include information on dosage, time, stress induction, etc. For example, a kit may include a tracer or precursor to the tracer described herein, together with instructions describing the desired application and proper administration of the agent to the subject. The term instructions, as used herein, may define a component of an instruction and / or promotion, and usually implies written instructions on the package of the invention or that are associated with it. The instructions may also include any oral or electronic format instructions provided in any way such that the user clearly acknowledges that the instructions should be associated with the kit, for example,

164 audiovisual communications (eg, videotape, DVE), internet and / or web. Written instructions may be in a prospectus prescribed by a government agency that regulates the manufacture, use or sale of pharmaceutical products, the instructions also reflecting the authorization of the agency for its manufacture, use or sale for administration purposes to beings humans. In some cases, the instructions may include instructions for mixing a particular amount of the diluent with a particular amount of a concentrated solution of the tracer or a solid preparation of the tracer, whereby a final formulation for injection or infusion is prepared such that, for example, the resulting solution has ^uriat a concentration suitable for administration to a subject (eg, a concentration described herein). A kit may include a complete treatment regimen of the compound of the invention.

The kit may contain one or more of any of the components described herein in one or more packages. By way of example, in one embodiment, the kit may include instructions for mixing one or more components of the kit and / or isolating and mixing a sample and applying it to a subject. The kit may include a package containing an agent described herein (eg, a precursor to the tracer or a tracer). The agent may be in the form of a liquid,

165 gel or solid (e.g., powder). The agent can be prepared under aseptic conditions, packaged in a syringe and sent under refrigeration conditions. Alternatively, it can be placed in a vial or other container for storage. A second container may contain other agents prepared under aseptic conditions. Alternatively, the kit may include a pre-mixed agent and sent in a syringe, vial, tube or other container. The kit may contain one or more or all of the components necessary to administer the agents to a subject, such as a syringe or a needle, tubes and an iv bag.

It will also be understood that packages containing the components of a kit of the invention, whether the container is a bottle, a vial (e.g., with a septum), a vial, an infusion bag or the like, may include additional indicators such as conventional markers that change color when the preparation has been sterilized. A kit of the invention may further include other components such as syringes, labels, vials, tubes, catheters, needles, entries and the like. In some aspect of the invention, a kit may include a single syringe containing sufficient tracing agent of the invention (e.g., tracer-1 agent) for administration and, in some aspects of the invention, a kit may include More than one syringe.

166

Buffers useful in the preparation of tracing agents and kits include, for example, phosphate, citrate, sulphosalicylate and acetate buffers. A more comprehensive list can be found in the United States Pharmacopoeia. Freeze-drying adjuvants useful in the preparation of tracing agents and kits include, for example, mannitol, lactose, sorbitol, dextran, FICOLL® polymer and polyvinylpyrrolidine (PVP). Stabilizing adjuvants useful in the preparation of tracing agents and kits include, for example, ascorbic acid, cysteine, monothioglycerol, sodium bisulfite, sodium metabisulfite, gentisic acid and inositol. Solubilization adjuvants useful in the preparation of tracing agents and kits include, for example, ethanol, glycerin, polyethylene glycol, propylene glycol, polyoxyethylene sorbitan monooleate, sorbitan monooleate, polysorbates, poly (oxyethylene) -poly (oxypropylene) block copolymers. -poli (oxyethylene) (e.g., Pluronics®) and lecithin. In certain embodiments, solubilization adjuvants are polyethylene glycol, cyclodextrins and Pluronics. Bactericides useful in the preparation of tracing agents and kits include, for example, benzyl alcohol, benzalkonium chloride, chlorbutanol and methyl-, propyl- or butylparaben. Definitions

For practical purposes, certain items are listed below.

167 terms used in the description, examples and appended claims.

Definitions of specific functional groups and chemical terms are described in more detail below. For the purposes of this invention, chemical elements are identified according to the periodic table of the elements, CAS version, Handbook of Chemistry and Physics, 75.^to ed. , inside cover, and specific functional groups are defined in general as described in the book. In addition, the general principles of organic chemistry, as well as reactivity and specific functional moieties, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito: 1999, whose

<td>content is</td><td>incorporates</td><td>in</td><td>fully to</td><td>present by</td>

<td>reference. Some</td><td>compounds</td><td>from</td><td>the present</td><td>invention can</td>

<td>exist in</td><td>shapes</td><td colspan="2">geometric or</td><td>stereoisomeric</td>

<td>private individuals</td><td colspan="2">The present</td><td colspan="2">invention encompasses all these</td>

<td>compounds,</td><td>included</td><td>the</td><td>cis isomers</td><td>and trans, the</td>

R and s enantiomers, diastereomers, D isomers, l isomers, racial mixtures of these and other mixtures thereof, within the scope of the invention. There may be additional asymmetric carbon atoms present in a substituent such as an alkyl group. It is intended that all these isomers, as well as mixtures thereof, remain

168 included in this invention.

In accordance with the present invention, isomeric mixtures containing any of various proportions of isomers can be employed. For example, when only two isomers are combined, mixtures containing an isomer ratio of 50:50, 60:40, 70:30, 80:20, 90:10, 95: 5, 96: 4, 97: 3 , 98: 2, 99: 1 or 100: 0 are all encompassed in the present invention. It will be obvious to those skilled in the art that analogous proportions will be encompassed for mixtures of more complex isomers.

In the event that, for example, a particular enantiomer of a compound of the present invention is desired, it may be prepared by asymmetric synthesis or by derivatization with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group is removed to provide the desired pure enantiomers. Alternatively, when the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts with a suitable optically active acid or base will be formed, then the resolution of the diastereomers formed in this way by fractional crystallization or chromatography, methods well known in the art, and then the pure enantiomers will be recovered.

At the term alkyl as used in the

169 present, it is given its usual meaning in the art and refers to the radical of saturated aliphatic groups, including straight chain alkyl groups, branched chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups and substituted alkyl groups with cycloalkyl. In some cases, the alkyl group may be a lower alkyl group, that is, an alkyl group having 1 to 10 carbon atoms (eg. , methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl or decyl). In some embodiments, a straight chain or branched chain alkyl may contain 30 or less carbon atoms in its backbone and, in some cases, 20 or less. In some embodiments, a straight chain or branched chain alkyl may contain 12 or less carbon atoms in its skeleton (e.g., Ci-C_i2 for the linear chain, C₃-Ci₂ for branched chain), 6 or less, or 4 or less. Similarly, cycloalkyls may contain 3-10 carbon atoms in their ring structure, or 5, 6 or 7 carbons in the ring structure. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, t-butyl, cyclobutyl, hexyl and cyclohexyl.

The terms alkenyl and alkynyl are given their usual meaning in the art and refer to unsaturated aliphatic groups analogous in length and possible

170 substitution to the alkyl described above, but containing at least one double or triple bond, respectively.

In certain embodiments, the alkyl, alkenyl and alkynyl groups employed in the invention contain 1-20 aliphatic carbon atoms. In certain embodiments, the alkyl, alkenyl and alkynyl groups used in the invention contain 1-10 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl and alkynyl groups used in the invention contain 1-8 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl and alkynyl groups used in the invention contain 1-6 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl and alkynyl groups used in the invention contain 1-4 carbon atoms. Thus, illustrative aliphatic groups include, but are not limited to, for example, methyl, ethyl, n-propyl, isopropyl, allyl, n-butyl, sec-butyl, isobutyl, t-butyl, n-pentyl, sec-pentyl , isopentyl, t-pentyl, n-hexyl, sec-hexyl, moieties and the like, which in turn may contain one or more substituents. Alkenyl groups include, but are not limited to, for example, ethenyl, propenyl, butenyl, 1-ethyl-2-buten-l-yl, and the like. Representative alkynyl groups include, but are not limited to, ethynyl, 2-propyl (propargyl), 1-propynyl and the like.

The term cycloalkyl, as used in the

171 present, it refers specifically to groups containing from three to ten, preferably from three to seven carbon atoms. Suitable cycloalkyls include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like, which, as in the case of other aliphatic, heteroaliphatic or heterocyclic moieties, may be optionally substituted with substituents that include, without limitation, aliphatic substituents; heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; -F; -Cl; -Br; -I; -OH; -DO NOT₂; -CN; -CF₃; -CH₂CF₃; -CHC1₂; -CH₂OH; -CH₂CH₂OH; -CH₂NH₂; CH₂SW₂CH₃; -C (O) R_x; -C0₂ (R_x); -CON (R_X)₂; -OC (O) R_X; -0CO₂R_x; OCON (R_x)₂; -N (R_x)₂; -SW)₂R_x; -NR_X(CO) R_Xwhere R_x in each case independently includes, without limitation, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl or heteroarylalkyl substituents, where any of the aliphatic, heteroaliphatic, arylalkyl or heteroarylalkyl substituents described above and may be substituted or unsubstituted herein, may be branched or unbranched, be cyclic or acyclic, and where any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Other examples of substituents

172 Generally applicable are illustrated in the specific modalities shown in the examples described herein.

The term "heteroalkyl" is given its usual meaning in the art and refers to an alkyl group described herein, in which one or more carbon atoms have been replaced by a heteroatom. Suitable heteroatoms include oxygen, sulfur, nitrogen, phosphorus and the like. Examples of heteroalkyl groups include, but are not limited to, alkoxy, amino, thioester, poly (ethylene glycol) and alkyl substituted amino.

The terms heteroalkenyl and heteroalkynyl are given their usual meaning in the art and refer to unsaturated aliphatic groups analogous in length and possible substitution to the heteroalkyl described above, but containing at least one double or triple bond, respectively.

Some examples of the substituents of the aliphatic moieties (and others) described above of the compounds of the invention include, without limitation, aliphatic substituents; heteroaliphatic; aryl, heteroaryl; alkylaryl; alkylheteroaryl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; C1; Br; I; -OH; -DO NOT₂; -CN; -CF₃; -CHF₂; -CH₂F; -CH₂CF₃; -CHC1₂; -CH₂OH; -CH₂CH₂OH; -CH₂NH₂;

173

-CH₂SW₂CH₃; -C (O) R_x; -CO₂(R_x); -CON (R_x)₂; -OC (0) R_x; -OCO₂R_x; OCON (R_x)₂; -N (R_x)₂; -SW)₂R_x; -NR_x(CO) R_xwhere R_x in each case independently includes, without limitation, aliphatic, alicyclic, heteroaliphatic, heterocyclic, aryl, heteroaryl, alkylaryl or alkylheteroaryl substituents, where any of the aliphatic, heteroaliphatic, alkylaryl or alkylheteroaryl substituents described above and in the present may be substituted or not substituted, may be branched or unbranched, be cyclic or acyclic, and where any of the aryl or heteroaryl substituents described above and herein may be substituted or unsubstituted. Other examples of substituents generally applicable are illustrated in the specific modalities shown in the examples described herein.

The term aryl is given its usual meaning in the art and refers to aromatic carbocyclic groups, optionally substituted, containing a single ring (e.g., phenyl), multiple rings (e.g., biphenyl) or multiple rings condensates in which at least one is aromatic (eg, 1,2,3,4-tetrahydronaphthyl, naphthyl, antrile or phenanthryl). That is, at least one ring may contain a conjugated pi electron system, while the other attached rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl and / or heterocyclyl.

174

The aryl group may be optionally substituted, as described herein. The substituents include, without limitation, any of the substituents mentioned above, that is, the substituents indicated for aliphatic moieties or for other moieties described herein that result in the formation of a stable compound. In some cases, an aryl group is a stable mono- or polycyclic unsaturated moiety that preferably contains 3-14 carbon atoms, each of which may be substituted or unsubstituted. The term "carbocyclic aryl groups" refers to aryl groups in which the ring atoms of the aromatic ring are carbon atoms. The carbocyclic aryl groups include monocyclic carbocyclic aryl groups and polycyclic or condensed compounds (e.g. eg, two or more adjacent ring atoms are

<td>common</td><td>in two</td><td>attached rings) such as</td><td>the groups</td>

<td>Naphthyl</td><td></td><td></td><td></td>

<td>To the</td><td>finished</td><td>heteroaryl is granted its</td><td>meaning</td>

<td>habitual</td><td>in the</td><td>technique and refers to groups</td><td>arilo that</td>

They comprise at least one heteroatom as the ring atom. A heteroaryl is a stable heterocyclic or polyheterocyclic unsaturated moiety that preferably contains 3-14 carbon atoms, each of which may be substituted or unsubstituted. Substituents include, without limitation, any of the substituents

175 mentioned above, that is, the substituents indicated for aliphatic moieties or for other moieties described herein that result in the formation of a stable compound. In some cases, a heteroaryl is a cyclic aromatic radical containing from five to ten ring atoms, of which one ring atom is selected from S, O and N; none, one or two ring atoms are additional heteroatoms independently selected from S, 0 and N; and the remaining ring atoms are carbon, the radical being attached to the rest of the molecule by any of the ring atoms, such as, for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl and the like.

It will also be understood that the aryl and heteroaryl moieties, as defined herein, can be linked by an alkyl or heteroalkyl moiety and, therefore, also include - (alkyl) aryl, - (heteroalkyl) aryl , - (heteroalkyl) heteroaryl and (heteroalkyl) heteroaryl. Therefore, the terms aryl or heteroaryl and aryl, heteroaryl, - (alkyl) aryl, - (heteroalkyl) aryl, (heteroalkyl) heteroaryl and - (heteroalkyl) heteroaryl moieties, as used herein, are interchangeable. The

176 Substituents include, without limitation, any of the substituents mentioned above, that is, the substituents indicated for aliphatic moieties or for other moieties described herein that result in the formation of a stable compound.

It should be noted that the aryl and heteroaryl groups (including bicyclic aryl groups) may be substituted or unsubstituted, where the substitution includes replacing one or more of the hydrogen atoms thereof independently with one or more of any of the following moieties, which they include, without limitation: aliphatic remains; alicyclics; heteroaliphatic; heterocyclic; aromatic; heteroaromatic; aryl; heteroaryl; alkylaryl; heteroalkylaryl; alkylheteroaryl; heteroalkyl heteroaryl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; C1; Br; I; -OH; -DO NOT₂; -CN; -CF₃; -CH₂F; -CHF₂; -CH₂CF₃; -CHC1₂; -CH₂OH; -CH₂CH₂OH; -CH₂NH₂; -CH₂SW₂CH₃; -C (O) R_x; -CO₂(R_x); -C0N (R_x)₂; -OC (O) R_X; -OCO₂R_x; OCON (R_x)₂; -N (R_x)₂; -S (O) R_x; -SW)₂R_x; -NR_x(CO) R_xwhere R_x in each case it includes independently, without limitation, aliphatic, alicyclic, heteroaliphatic, heterocyclic, aromatic, heteroaromatic, aryl, heteroaryl, alkylaryl, alkylheteroaryl, heteroalkylaryl or heteroalkyl heteroaryl substituents, where any

177 of the aliphatic, alicyclic, heteroaliphatic, heterocyclic, alkylaryl or alkylheteroaryl substituents described above and herein may be substituted or unsubstituted, branched or unbranched, saturated or unsaturated, and where any of the aromatic, heteroaromatic, aryl, heteroaryl substituents, - (alkyl) aryl or - (alkyl) heteroaryl described above and herein may be substituted or unsubstituted. In addition, it should be noted that any two adjacent groups considered together may represent a substituted or unsubstituted heterocyclic or alicyclic moiety of 4, 5, 6 or 7 members. Other examples of substituents generally applicable are illustrated in the specific modalities described herein.

The term heterocycle is given its usual meaning in the art and refers to cyclic groups that contain at least one heteroatom as the ring atom, in some cases, 1 to 3 heteroatoms as ring atoms, the rest of the ring atoms being atoms of carbon. Suitable heteroatoms include oxygen, sulfur, nitrogen, phosphorus and the like. In some cases, the heterocycle may be a 3 to 10 member ring structure or a 3 to 7 member ring, whose ring structure includes one to four heteroatoms.

The term heterocycle may include groups

178 heteroaryl, saturated heterocycle groups (eg, cycloheteroalkyl) or combinations thereof. The heterocycle may be a saturated molecule or it may comprise one or more double bonds. In some cases, the heterocycle is a heterocycle with nitrogen, where at least one ring comprises at least one annular nitrogen atom. Heterocycles can be condensed with other rings to form a polycyclic heterocycle. The heterocycle can also be condensed with a spirocyclic group. In some cases, the heterocycle can be attached to a compound through a nitrogen or carbon atom in the ring.

Heterocycles include, for example, thiophene, benzothiophene, thiantrene, furan, tetrahydrofuran, pyran, isobenzofuran, chromene, xanthene, phenoxyatin, pyrrole, dihydropyrrole, pyrrolidine, imidazole, pyrazole, pyrazine, isothiazole, isoxazole, pyridine, pyrazine, pyrazine , indolizine, rsoindole, indole, indazole, purins, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinoline, pteridine, carbazole, carboline, triazole, tetrazole, oxazole, isoxazole, thiazole, isothiazole, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, oxazine, piperidine, homopiperidine (hexamethyleneimine), piperazine (p. g., N-methylpiperazine), morpholine , lactones, lactams such as

179 azetidinones and pyrrolidinones, sultamas, sultones, other saturated and / or unsaturated derivatives thereof, and the like. The heterocyclic ring may be optionally substituted in one or more positions with substituents such as those described herein. In some cases, the heterocycle can be linked to a compound through an annular atom that is a heteroatom (eg, nitrogen). In some cases, the heterocycle can be linked to a compound through an annular atom that is carbon. In some cases, the heterocycle is pyridine, imidazole, pyrazine, pyrimidine, pyridazine, acridine, acridin-9-amine, bipyridine, naphthyridine, quinoline, benzoquinoline, benzoisoquinoline, phenanthridine-1,9-diamine or the like.

The terms halo and halogen, as used herein, refer to an atom selected from fluorine, chlorine, bromine and iodine.

The term "haloalkyl" refers to an alkyl group, as defined above, which contains one, two or three halogen atoms attached thereto; Some examples are groups such as chloromethyl, bromoethyl, trifluoromethyl and the like.

The term amino¹¹, as used herein, refers to a primary (-NH2), secondary (-NHRX), tertiary (-NRxRy) or quaternary (-N⁺RxR_YR_z), where R_x, R_Y and R_zthey are independently an aliphatic, alicyclic moiety,

180 heteroaliphatic, heterocyclic, aryl or heteroaryl as defined herein. Examples of amino groups include, but are not limited to, methylamino, dimethylamino, ethylamino, diethylamino, methylethylamino, isopropylamino, piperidino, trimethylamino and propylamino.

The term "alkyne" is given its usual meaning in the art and refers to branched or unbranched unsaturated hydrocarbon groups containing at least one triple bond. Non-limiting examples of alkynes include acetylene, propino, 1-butyne, 2-butyne and the like. The alkyne group may be substituted and / or one or more of its hydrogen atoms may have been replaced by a functional group such as a hydroxyl, halogen, alkoxy and / or aryl group.

The term "alkoxy" (or alkyloxy) or thioalkyl, as used herein, refers to an alkyl group, as previously defined, attached to the original molecular moiety by an oxygen atom or by a sulfur atom. In certain embodiments, the alkyl group contains 120 aliphatic carbon atoms. In certain other embodiments, the alkyl group contains 1-10 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl and alkynyl groups used in the invention contain 1-8 aliphatic carbon atoms. In still other embodiments, the alkyl group contains 1-6 aliphatic carbon atoms. In others

181 further embodiments, the alkyl group contains 1-4 aliphatic carbon atoms. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, t-butoxy, neopentoxy and n-hexoxy. Examples of thioalkyl include, but are not limited to, methylthio, ethylthio, propylthio, isopropylthio, n-butylthio and the like.

The term "aryloxy" refers to the group -0-aryl. The term "acyloxy" refers to the group -O-acyl.

The term "alkoxyalkyl" refers to an alkyl group substituted with at least one alkoxy group (eg, one, two, three or more alkoxy groups). For example, an alkoxyalkyl group may be - (C ^ alkyl) - 0- (alkyl, optionally substituted. In some cases, the alkoxyalkyl group may be optionally substituted with another alkoxyalkyl group (eg, - (C ^ alkyl) -0- (CYJ-O alkyl (C! Alkyl.₆), optionally substituted.

It should be noted that the above groups and / or compounds described herein may be optionally substituted with any number of substituents or functional moieties. That is, any of the above groups may be optionally substituted. It is intended that the substituted term, as used herein, includes all permitted substituents of organic compounds, the term allowed being adjusted in the context of chemical standards concerning valence with

182 those who will be familiar to those skilled in the art. In general, the term substituted, whether preceded by the term optionally or not, and the substituents contained in the formulas of this invention, refer to the replacement of hydrogen radicals in a structure determined by the radical of a specified substituent. When more than one position in a given structure can be substituted with more than one substituent selected from a specified group, the substituent may be the same or different in each position. It will be understood that the term substituted also includes the fact that the substitution results in a stable compound, e.g. eg, that does not spontaneously undergo a transformation such as transposition, cyclization, elimination, etc. In some cases, the term substituted may in general refer to the replacement of a hydrogen with a substituent, as described herein. However, the term substituted, as used herein, does not encompass the replacement or alteration of a key functional group with which a molecule is identified, e.g. eg, so that the substituted functional group becomes, through substitution, a different functional group. For example, a substituted phenyl group must continue to comprise the phenyl moiety and cannot be modified by substitution, in this definition, to be converted, e.g. eg, in a ring of

183 pyridine In a broad aspect, the permitted substituents include cyclic and acyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein. The substituents allowed may be one or more, and may be the same or different for suitable organic compounds. For the purposes of this invention, heteroatoms, such as nitrogen, may have substituents that are hydrogen and / or any permitted substituent of organic compounds described herein that meets the valencies of the heteroatoms. Furthermore, it is not intended that this invention be limited in any way by the permitted substituents of the organic compounds. The combinations of substituents and variables that are considered in this invention are preferably those that result in the formation of stable compounds useful for the formation of a tracer or precursor of the tracer. The term "stable", as used herein, preferably refers to compounds that have sufficient stability for production to be possible and that maintain the integrity of the compound for a period of time sufficient for them to be detected and preferably for a period of sufficient time for them to be useful for the purposes detailed herein.

184

Examples of substituents include, but are not limited to, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxy, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, cebone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF₃, -CN, aryl, aryloxy, perhaloalkoxy, aralkoxy, heteroaryl, heteroaryloxy, heteroarylalkyl, heteroaralkoxy, azido, amino, halide, alkylthio, oxo, acylalkyl, carboxy esters, -carboxamido, acyloxy, aminoalkyl, alkylaminoarylalkyl, alkylarylalkyl, alkylarylalkyl, alkylaryloxylalkyl, alkylarylalkyl, alkylarylalkyl arylamino, aralkylamino, alkylsulfonyl, -carboxamidoalkylaryl, -carboxamidoaryl, hydroxyalkyl, haloalkyl, alkylaminoalkylcarboxy-, aminocarboxamidoalkyl-, cyano, alkoxyalkyl, perhaloalkyl, arylalkyloxyalkyl and the like.

The term "determine", as used herein, refers generally to the analysis of a species or signal, for example, qualitatively or quantitatively, and / or to the detection of the presence or absence of the species or signal.

The term "diagnostic tomography," as used herein, refers to a procedure used to detect a tracing agent.

The term diagnosis, as used herein, encompasses identification, confirmation and / or

185 characterization of a condition, a disease and / or a disorder.

A diagnostic kit or kit comprises a collection of components, called the formulation, in one or more vials, which the end user uses for practical purposes in a clinical or pharmaceutical environment to synthesize diagnostic radiopharmaceutical agents. For example, the kit can be used by the end user for practical purposes in a clinical or pharmaceutical environment to synthesize and / or use diagnostic radiopharmaceutical agents. In some embodiments, the kit can provide all the necessary components to synthesize and use the pharmaceutical diagnostic agent, except for those usually available to the end user for practical purposes, such as water or saline for injection, and / or the radioisotope (e.g., ^iaF), the equipment to process the kit during the synthesis and manipulation of the radiopharmaceutical agent, if appropriate, the equipment necessary to administer the radiopharmaceutical agent to the subject such as syringes, protection, tomographic equipment and the like. In some embodiments, the tracing agents can be provided to the end user in their final form in a formulation usually contained in a vial or syringe, either as a lyophilized solid or as an aqueous solution.

A portion of a subject, as used herein, refers to a particular region of a subject, a

186 Place in the subject. For example, a portion of a subject may be the brain, the heart, the vasculature, the cardiac vessels, etc. of a subject.

A test session, as used herein, may be a single test protocol to which a subject is subjected.

The term "subject", as used herein, refers to a human being, or a non-human animal or mammal. Non-human mammals include livestock, companion animals, laboratory animals and non-human primates. Non-human subjects also specifically include, without limitation, horses, cows, pigs, goats, dogs, cats, mice, rats, guinea pigs, Gerbils, hamsters, minks and rabbits. In some embodiments of the invention, the subject is called a patient. In some modalities, a doctor or other healthcare professional may be in charge of a patient or subject including, but not limited to, someone who has attended a consultation, someone who has received advice or who has received a prescription or other recommendation from a doctor or other healthcare professional.

Any of the compounds described herein can take various forms such as, without limitation, salts, solvates, hydrates, tautomers and isomers.

In certain embodiments, the tracing agent is a salt.

187 pharmaceutically acceptable tracer agent. The term "pharmaceutically acceptable salt", as used herein, refers to those salts that are, within the scope of reasonable medical criteria, suitable for use in contact with the tissues of humans and lower animals without causing toxicity, irritation, allergic and similar responses that are excessive, and correspond to a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutically acceptable salts

<td>with</td><td>detail in J.</td><td>Pharmaceutical</td><td>Sciences, 1977,</td><td> 66,</td><td> 1-19,</td>

<td>what</td><td>is incorporated</td><td>to the present</td><td>by reference</td><td>The</td><td>you go out</td>

<td colspan="2">pharmaceutically</td><td>acceptable from</td><td>the compounds</td><td>from</td><td>is</td>

Invention include those that are derived from suitable organic and inorganic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of an amino group formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or with organic acids, such as acetic acid, acid. oxalic, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid, or using other methods that are used in the art such as ion exchange. Other salts pharmaceutically

188 Acceptable include salts of adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, canforate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucohepatoate, heptaphonate, heptaphonate, heptane hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate and the like. Salts derived from suitable bases include alkali metal, alkaline earth metal, ammonium and N salts.⁺(alkyl 0 ^ 4) 4. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium and the like. Other pharmaceutically acceptable salts include, where appropriate, non-toxic cations of ammonium, quaternary ammonium and amine, formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, (lower alkyl) sulfonate and arylsulfonate.

In certain embodiments, the compound takes the form

189 of a hydrate or solvate. The term "hydrate", as used herein, refers to a compound that is not covalently associated with one or more water molecules. Similarly, the term "solvate" refers to a compound that is not covalently associated with one or more molecules of an organic solvent.

In certain embodiments, the compound described herein may exist in various tautomeric forms. The term tautomer, as used herein, includes two or more compounds that can be interconverted as a result of at least one formal migration of a hydrogen atom and at least one change of valence (e.g., of a bond single to a double link, from a triple link to a single link or vice versa). The exact proportion of tautomers depends on several factors that include temperature, solvent and pH. The tautomerizations (ie, the reaction that provides a tautomeric pair) can be catalyzed with an acid or a base. Examples of tautomerizations include the tautomerization of ketone to enol, amide to imide, lactam to lactime, enamine to imine and enamine to a different enamine.

In certain embodiments, the compounds described herein may exist in several isomeric forms. The term isomer, as used herein, includes each and every one of the geometric isomers and

190 stereoisomers (e.g., enantiomers, diastereomers, etc.). For example, the term isomer includes cis and trans isomers, E and Z isomers, R and S enantiomers, diastereomers, d isomers, l isomers, their racemic mixtures and other mixtures thereof, within the scope of the invention. For example, in some embodiments, an isomer / enantiomer can be provided substantially free of the corresponding enantiomer and can also be said to be optically enriched. The term optically enriched, as used herein, refers to the compound being constituted by a significantly greater proportion of an enantiomer. In certain embodiments, the compound of the present invention is constituted by at least about 9% by weight of a preferred enantiomer. In other embodiments, the compound is constituted by at least about 95%, 9B% or 99% by weight of a preferred enantiomer. Preferred enantiomers can be isolated from racemic mixtures by any method known to those skilled in the art that includes chiral high resolution liquid chromatography (HPLC) and the formation and crystallization of chiral salts or prepared by asymmetric synthesis. Refer, for example, to Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York,

191

nineteen eighty one); Wilen, SH et al., Tetrahedron 33: 2725 (1977);

Eliel, EL Stereochemistry of Carbon Compounds (McGrawHill, NY, 1962); Wilen, SH Tables of Resolving Agents and Optical Resolutions p. 268 (El Eliel, Ed., Univ. Of Notre Dame Press, Notre Dame, IN 1972).

These and other aspects of the present invention will be more clearly appreciated taking into account the following examples, which are intended to illustrate certain particular embodiments of the invention, but are not intended to limit its scope, as defined in the claims.

Examples

Example 1

Synthesis of 3- (4 - ((1, 2-bis (tertbutoxycarbonyl) guanidino) methyl) -2-bromophenoxy) propyl 4-methylbenzenesulfonate

NBoc

NH₂

Boc

N and Boc

Example 1A

Synthesis of 1,2-bis (tert-butoxycarbonyl) -1- [3-bromo-4- (3hydroxypropoxy) benzyl] guanidine

BrNBoc

192

To a solution of 1,2-bis (tert-butoxycarbonyl) -1- [3bromo-4-hydroxybenzyl] guanidine (for synthesis, see, for example, Purohit et al., PCT International Patent Publication No. W02008 / 083056, incorporated herein by reference) (2.0 g, 4.51 mmol) dissolved in DMF (45 mL) K were added₂CO₃ (1.12 g, 8.13 mmol) and 3bromopropanol (816 mg, 5.87 mmol), and the reaction mixture was heated to 50 ° C with an oil bath. After 2 h, the reaction mixture was diluted with water (30 mL), the aqueous layer was separated and then extracted with EtOAc (3 x 100 mL). The combined organic layers were dried with MgSO₄, filtered and concentrated to obtain a solid. The crude material was purified by silica gel chromatography (from 4: 1 to 3: 2 hexanes: EtOAc) to obtain a white solid product (2.00 g, 88% yield).^rH NMR (CDC13, 600 MHz): δ 9.42 (sa, 1H), 9.27 (sa, 1H), 7.54 (d, J = 1.8 Hz, 1H), 7.26 (m, 1H), 6.85 (d, J = 2.4 Hz, 1H), 5.08 (sa, 2H), 4.19 (t, J = 5.4 Hz, 2H), 3.92 (m, 2H), 2.16 (m, 1H), 2.18 (m, 2H), 1.51 (s, 9H ), 1.43 (s, 9H); ¹³NMR C (CDC13, 150 MHz): δ 163.8, 160.8, 155.0, 154.3, 144.8, 133.1, 132.6,

127.9, 113.0, 111.7, 84.7, 79.2, 67.8, 60.6, 46.7, 31.9,

28.5, 28.3. Example IB Synthesis of 3- (4 - ((1,2bis (tert-butoxycarbonyl) guanidino) methyl) -2 193 bromophenoxy) propyl 4-methibenzenesulfonate

NBoc

I'

<img file="MX367382B_D0087.tif" />

'σ

<img file="MX367382B_D0088.tif" />

NH₂

Boc

To a solution of the product of Example 1A (339 mg, 0.676 immoles) dissolved in CH₂C1₂ anhydrous (6.76 mL), TsCl (155 mg, 0.812 mmol), DMAP (99 mg, 0.812 mmol) and Et were added₃N (0.141 mL, 1.01 mmol). The reaction mixture was stirred at room temperature for 1.5 h and then concentrated to obtain a yellow oil. The crude material was purified directly by silica gel chromatography (4: 1 hexanes: EtOAc) to obtain a colorless oil (384.3 mg, 87% yield).³H NMR (CDC13, 600 MHz): δ 7.74 (d, &lt;7 = 8.4 Hz, 2H), 7.50 (d, &lt;7 = 1.8 Hz, 1H), 7.21 (m, 3H), 6.70 (d, &lt;7 = 8.4 Hz, 1H), 5.08 (sa, 2H), 4.30 (t, &lt;7 = 6.0 Hz, 2H), 4.00 (t, &lt;7 = 6.0 Hz, 2H), 2.37 (s, 3H), 2.16 (m, 2H), 1.51 (s, 9H), 1.43 (s, 9H); ¹³NMR C (CDC13, 150 MHz): δ 160.6, 154.9, 154.0, 145.0, 133.0, 132.9, 132.7, 130.0, 128.0, 112.9, 111.9, 84.7, 79.0, 67.0, 64.1, 46.4, 29.0, 28.5, 28.2, 21.8. Example 2

Synthesis of 3- (4 - ((1,2 bis (tert-butoxycarbonyl) guanidino) methyl) -2194 brornophenoxy) propyl 4-bromobenzenesulfonate

NBoc

<img file="MX367382B_D0089.tif" />

To a solution of the product of Example 1A (300 mg, 0.598 immoles) dissolved in CH₂C1₂ anhydrous (6.0 mL), BsCl (183.3 mg, 0.718 mmol), DMAP (87.7 mg, 0.718 mmol) and Et was added₃N (0.125 mL, 0.897 mmol). The reaction mixture was stirred at room temperature for 2.5 h and then concentrated to obtain an oil. The crude material was purified directly by silica gel chromatography (4: 1 hexanes: EtOAc) to obtain a colorless oil (395.6 mg, 92% yield).^XNMR H (CDC13, 300 MHz): 59.40 (sa, 2H), 7.72-7.67 (m, 2H), 7.55-7.50 (m, 3H), 7.24 (dd, J = 3, 9 Hz, 1H), 6.69 ( d, J = 9 Hz, 1H), 5.11 (sa, 2H), 4.35 (t, J = 6.0 Hz, 2H), 3.97 (t, J = 6.0 Hz, 2H), 2.18 (m, 2H), 1.47 ( s, 9H), 1.39 (s, 9H); ¹³NMR C (4: 1, CDC13: DMSO-d₆ ,150

MHz): 5160.7, 160.5, 157.1, 153.5, 134.0, 132.0, 131.6, 130.3, 130.2, 128.6, 128.3, 127.2, 127.2, 112.4, 111.3,

84.5, 79.0, 66.8, 63.4, 42.3, 27.4. Example 3 Synthesis of 3 - (4 - ((1,2-bis (tert195 butoxycarbonyl) guanidine) methyl) -2-bromophenoxy) propyl methanesulfonate

NBoc

Boc

To a solution of the product of Example 1A (300 mg, 0.598 mmol) dissolved in CH₂C1₂ anhydrous (6.0 mL), MsCl (55.8 μΐι, 0.718 mmol), DMAP (87.7 mg, 0.718 mmol) and Et were added₃N (0.125 mL, 0.897 mmol). The reaction mixture was stirred at room temperature for 45 min and then concentrated to obtain an oil. The crude material was purified directly by silica gel chromatography (4: 1 hexanes: EtOAc) to obtain a colorless oil (245.6 mg, 71% yield).^ΣΗ NMR (CDClj, 300 MHz): δ 9.35 (sa, 2H), 7.56 (d, &lt;7 = 3.0 Hz, 1H), 7.26 (m, 1H). 6.84 (d, &lt;7 = 9.0 Hz, 1H), 5.09 (sa, 2H), 4.53 (t, &lt;7 = 6.0

Hz, 2H), 4.15 (t, &lt;7 = 6.0 Hz, 2H), 3.01 (s, 3H), 2.29 (m, 2H), 1.52 (s, 9H), 1.43 (s, 9H); ¹³C (CDC1₃, 150 MHz): δ 160.7, 154.9, 154.1, 133.3, 133.1, 128.0, 132.2, 113.2, 110.7, 128.3, 84.7, 80.5, 66.9, 64.6, 46.7, 29.9, 28.5, 28.2. Example 4

Synthesis of 3- (4- ((1,2-bis (tertbutoxycarbonyl) guanidino) met i1) -2-bromophenoxy) propyl trifluoromethanesulfonate

NBoc

<img file="MX367382B_D0090.tif" />

To a solution of the product of Example 1A (300 mg,

196 ίο

0.598 mmol) dissolved in CH₂C1₂ anhydrous (6.0 mL), Tf was added₂O (203 mg, 0.718 mmol), DMAP (87.7 tng, 0.718 mmol) and Et₃N (0.125 mL, 0.897 mmol). The reaction mixture was stirred at room temperature for 1.5 h and then concentrated to obtain an oil. The crude material was purified directly by silica gel chromatography (from 4: 1 to 1: 1 hexanes: EtOAc) to obtain a colorless oil (312 mg, 82% yield).³H NMR (CDC13, 300 MHz): δ 9.39 (sa, 2H), 7.54 (d, J = 3. 0 Hz, 1H), 7.26 (m, 1H), 6.84 (d, J = 9.0 Hz, 1H), 5.08 (sa, 2H), 4.16 (t, &lt;7 = 6.0 Hz, 2H), 3.81 (t, &lt;7 = 6.0 Hz, 2H), 2.27 (m, 2H), 1.50 (s, 9H), 1.39 (s , 9H); ¹³NMR C (CDC13, 150 MHz): δ 160.7, 154.9, 154.3, 133.2, 132.8, 128.1, 113.2, 112.0, 84.7, 79.3, 65.8, 46.7, 40.7, 32.4, 28.5, 28.2; ¹⁹F NMR (CDC1₃, 282 MHz): δ -75.5 (s).

Example 5

The following example describes the synthesis of compounds of formula (II) including, without limitation, the precursor of the tracer-1 agent. The example more specifically provides the synthesis of the trifluoroacetic acid salt of the tracer agent1 precursor, according to the scheme shown in Figure 6.

Example 5A

Synthesis of 3-Bromo-4- (3-hydroxypropoxy) benzonitrile

197 (Compound 1)

Brx ^ -CN

3-Bromo-4-hydroxybenzonitrile (10.0 g, 50.5 mmol) was dissolved in acetone and treated successively with l-bromo-3propanol (19.0 g, 138 mmol) and K₂CO₃ (20.9 g, 151 mmol) at room temperature. The resulting suspension was heated to 50 ° C and maintained at this temperature for 3 d. After cooling to room temperature, the solids were removed by filtration, washed thoroughly with acetone and the filtrate was concentrated. After purification by chromatography on SiO₂ (A: hexanes; B: EtOAc; 0-100% of B in 35.4 min; 200 mL / min; 330 g column), a solid was obtained. It was further purified by recrystallization from hot MTBE (131 mL) and pentane (130 mL) with cooling at -20 C (12 h) to induce precipitation and a solid (7.2 g, 58%) was obtained. NMR (300 MHz, CDC1₃) δ 7.78 (s, 1H), 7.56 (d, J = 9 Hz, 1H), 6.94 (d, J = 6 Hz, 1H), 4.23 (t, J = 6 Hz, 2H), 3.88 (t, J = 6 Hz, 2H), 2.08 (m, J = 6 Hz, 2H). Example 5A-1

The following example describes the synthesis of Compound 1, using a synthetic method alternative to Example 5A. 3-Bromo-4-hydroxybenzonitrile (0.100 kg, 0.505 mol) was added to a reaction vessel followed by 2-butanone (1.00 L), 3-chloro-1-propanol (50 mL, 0.598 mol), Na₂CO₃ (80.6 g, 0.760

198 i £ i.

mol) and Nal (15.0 g, 0.100 mol). Then, the reaction mixture was protected from light with aluminum foil, heated to reflux and stirred overnight. After 23 h, unreacted starting material was still present. Then, more 3-chloro-1-propanol (8.7 mL ·, 0.10 mol) was added and the mixture was reheated to reflux. After a total reflux time of 34 h, the heat was removed and the vessel was cooled slowly in 19 h to 22.8 ° C before adding MTBE (1.00 L). The resulting solution was stirred for 44 min, then a class C sintered glass funnel containing a 5 cm Celite bed was filtered through. The reaction vessel and the Celite bed were rinsed with several small portions of MTBE, and the combined filtrates were concentrated in vacuo.

The crude solid was dissolved in MTBE at reflux (410 mL) and then treated with heptane (410 mL) in 14 min to form an oil. Once the addition was finished, the heating blanket was removed and the biphase was cooled to 29.9 'C. After 1 h, the resulting suspension was diluted with heptane (1.18 L), stirred 56 min and then filtered through a class C sintered glass funnel. The solids were washed with 9: 1 heptane: MTBE (398 mL) and subsequently transferred to a drying tray and placed in a vacuum oven. After drying at 35 + 5 ° C

199 for 36 h, 118.4 g of the solid were obtained (0.462 mol; 91.5%).

Example 5B

Synthesis of 3-Bromo-4- (3-hydroxypropoxy) benzylamine hydrochloride (Compound 2)

NH₂ HCI

0'

Compound 1 (5.0 g, 19.5 minols) was suspended in THF, then stirred at room temperature until complete dissolution was observed. Then, BHj-THF (42.9 mmol; 42.9 mL of a 1.0 M solution in THF) was added dropwise and the resulting mixture was heated to reflux. After 5 h, the mixture was cooled to 4 'C and carefully treated with MeOH (50 mL). HC1 (g) was bubbled into the solution for 30 min and then all volatiles were removed in vacuo. The white solid obtained was dissolved in MeOH (17.8 mL), and then treated successively with MTBE (36 mL) and hexanes (40 mL). The resulting suspension was stirred for 30 min and the separated white solids were then dried to obtain a constant weight (4.7 g, 81%). This material was used directly in the next step without further purification. Example 5B-1

The following example disarms the synthesis of the Compound

200

2, using a synthetic method alternative to Example 5B. Compound 1 (118.4 g, 0.462 mol) was transferred, under a nitrogen atmosphere, to a reaction vessel together with anhydrous THF (1.16 L). The mixture was stirred until complete dissolution was observed and then slowly treated with BH.₃-THF (1.02 mol; 1.02 L of a 1.0 M solution in THF) in 20 min. Upon completion of the addition, the reaction vessel was heated to reflux and maintained at this temperature overnight. Subsequently, the resulting suspension was cooled to 29.9 ° C before applying an ice water bath to further reduce the internal temperature to 4.9 ° C. Then, hydrochloric acid (1.25 mol; 1.00 L of a 1.25 M solution in MeOH) was added dropwise in 94 min; a measured value of pH 3 confirmed the complete hydrolysis of the intermediate boronate species. Subsequently, the resulting mixture was concentrated to dryness in vacuo (&lt;35 ° C) to obtain a solid (172.1 g).

The crude product was transferred to a clean, new reaction vessel together with MeOH (279 mL). After stirring 20 min, the resulting suspension was treated with MTBE (550 mL ·), stirred 16 min and then diluted with heptane (1.10 L ·). After 2.5 h, the solids were isolated by filtration through a class C sintered glass funnel and then washed with 1: 1 heptane: MTBE (410 mL) before transferring them to a vacuum oven. After drying at 35+

201 ° C for 10 h, 119.2 g of a solid material were obtained.

Example 5C

Synthesis of l, 3-bis (tert-butoxycarbonyl) - [3-bromo-4- (3-hydroxypropoxy) benzyl] guanidine (Compound 3)

Br.

N NHBoc H

Compound 2 (0.438 g, 1.48 minols) was dissolved in MeOH (7.00 mL) and treated successively with N, Ν '-bis-tertbutoxycarbonyl-lH-pyrazolecarboxamidine (0.412 g, 1.33 mmol) θ i-Pr₂NEt (0.380 g, 2.95 mmol) at room temperature. The resulting mixture was stirred 3 h, then concentrated and purified by SiO chromatography.₂ (A: hexanes; B: EtOAc; 0-100% B in 19.2 min; 40 mL / min; 40 g column) to obtain the product as a white foam (0.61 g, 82%). ^XH NMR (300 MHz, CDC1₃) δ 8.5 (t, 1H), 7.5 (d, 1H), 7.2 (dd, 1H), 6.85 (d, 1H), 4.52 (d, 2H), 4.18 (t, 2H), 3.9 (t, 2H) , 2.1 (m, 2H), 1.52 (s, 9H), 1.47s (s, 9H). Example 5C-1

The following example describes the synthesis of Compound 3, using a synthetic method alternative to Example 5C. Compound 2 (119.1 g, 0.401 mol) was transferred to a reaction vessel with MeOH (1.13 L), N, Ν '-bis-tertbutoxycarbonyl-lH-pyrazolcarboxamidine (126.4 g, 0.408 mol) and i-Pr₂NEt (82.0 mL, 0.461 mol). The resulting mixture was stirred.

202 at room temperature for 13 h, then it was treated with EtOAc (150 ml ·) and concentrated to dryness in vacuo (305.7 g). The crude oil obtained in this way was transferred to a separatory funnel using 1.31 L of EtOAc and then washed with deionized water (417 mL). The aqueous layer was further washed with EtOAc (6 00 mL), and the combined organic layers were washed successively with 307 mL of NaHSO₄-H₂O 0.5 M, 300 mL of deionized water and 300 mL of NaHC0₃ 0.5 M, then dried with Na₂SW₄ in excess. The desiccant was removed by filtration through a class C sintered glass funnel and then washed with EtOAc (190 mL). The combined filtrates were concentrated in vacuo to obtain a light brown viscous oil (213 g).

Example 5D

Synthesis of 3 - (4 - ((2,3-bis (tertbutoxycarbonyl) guanidino) methyl) -2-bromophenoxy) propyl 4-bromobenzenesulfonate (Compound 4)

<img file="MX367382B_D0091.tif" />

NBoc

Compound 3 (0.2 g, 0.4 mmol) was treated successively with 4-bromobenzenesulfonyl chloride (173 mg, 0.677 mmol), Et₃N (80.62 mg, 0.796 mmol), DMAP (4.86 mg, 3.98 gmol) and CH₂C1₂ (8 mL) at room temperature. The resulting solution was stirred 24 h and then all

203 Vacuum volatile. The residue was washed with hexanes: EtOAc (10 mL ·; 9: 1 v / v) to obtain a white solid that was filtered off. After purification by chromatography on SiO₂ (TO:

hexanes; B: EtOAc; 0-100% of B in 15.4 min; 35 mL / min; 24 g column), the product was obtained as a sticky white solid (174 mg, 60%).^XH NMR (300 MHz, CDC1₃) δ 8.53 (t, 1H), 7.66 (m, 2H), Ί .5 (m, 3H), 7.17 (m, 1H), 6.67 (d, &lt;7 = 8.4 Hz, 1H), 4.53 (d, &lt; 7 = 5 Hz, 2H), 4.33 (t, &lt;7 = 6 Hz, 2H), 3.93 (t, &lt;7 = 6 Hz, 2H), 2.16 (m, 2H), 1.53 (s, 9H), 1.47 ( s, 9H). Example 5D-1

The following example describes the synthesis of Compound 4, using an alternative synthetic method with respect to Example 5D. Compound 3 (212.9 g, 0.424) was transferred to a reaction vessel, under a nitrogen atmosphere, using CH₂C1₂ anhydrous (2.00 L) and then stirred for 15 min until complete dissolution occurred. The resulting solution was treated successively with 4-bromobenzenesulfonyl chloride (125.5 g, 0.491 mol), Et₃N (80.0 mL, 0.573 mol) and DMAP (2.06 g, 0.017 mol), and then stirred vigorously for 16 hours at room temperature. NOTE: the process was relatively exothermic since the internal temperature reached 33.9 'C after adding the DMAP. Subsequently, more 4-bromobenzenesulfonyl chloride (10.5 g, 0.041 mol) was added and the resulting mixture was stirred 19 h. This process is

204 repeated once more using more 4bromobenzenesulfonyl chloride (20.9 g, 0.082 mol) and Et₃N (11.3 mL, 0.081 mol), and then stirred vigorously for 8 hours at room temperature. Then, the resulting solution was treated with deionized water (600 mL ·) and transferred to a separatory funnel. Subsequently, the layers were separated and the aqueous layer was washed with CH2CI2 (290 mL). The combined organic layers were subsequently washed with NaHCO₃ 5% aqueous (380 mL ·), dried with Na₂SW₄ in excess, and then filtered and concentrated in vacuo. The crude product was partially purified by silica gel chromatography (-20 g SiO₂/ g of crude product) using 10-20% EtOAc / heptane; similar fractions were combined and concentrated to obtain a solid in vacuo. The crude material obtained in this way was further purified by washing with MTBE (804 mL) and heptane (1580 mL), then isolated by filtration through a class C sintered glass funnel. The retained mass in the filter was washed with 9: 1 heptane / MTBE (467 mL ·), then transferred to a vacuum oven and dried 15 h at room temperature (149.4 g, 0.207 mol; 48.9%).

Example 5E Synthesis of 3- (2-Bromo-4 (guanidinomethyl) phenoxy) propyl 4-bromobenzenesulfonate, trifluoroacetic salt (salt of

205

TFA of the tracer agent precursor-1)

<img file="MX367382B_D0092.tif" />

Compound 4 (3.00 g, 4.15 minols) was introduced into a 25 mL round bottom flask followed by CH₂C1₂ (6 mL), and the resulting suspension was stirred until its complete dissolution was observed. Then, trifluoroacetic acid (6 mL, 78.3 immoles) was added and the mixture was stirred an additional 4 h. Subsequently, all volatiles were removed and the residue was treated with EtOAc (20 mL). The resulting mixture was stirred at room temperature for 3 h and during the period a white solid precipitated. The solids were separated with a sintered glass funnel of medium porosity, and then thoroughly washed with EtOAc (20 mL) and dried to obtain a constant weight (2.5 g, 95¾). NMR (400 MHz, DMSO-d_and) δ 7.54 (m, 4H), 7.4 (d, 1H), 7.15 (m, 1H), 6.9 (d, 1H), 4.15 (m, 4H), 3.86 (m, 2H), 1.92 (m, 2H) . Example 5E-1

The following example describes the synthesis of the TFA salt of the tracer-1 precursor, using an alternative synthetic method with respect to Example SE. Compound 4 (149.4 g, 0.207 mol) was dissolved in CH₂C1₂ (1.20 L) and then treated with TFA (300 mL) in a portion at room temperature. After 14 h, all were removed

206 The volatiles in vacuo and the crude oil were treated directly with EtOAc (1.32 L). After 3 h, the resulting suspension was filtered through a class C sintered glass funnel and the solids washed with EtOAc (2 x 140 mL). The mass retained in the filter was then transferred to a glass drying tray and placed in a vacuum oven for 12 h at room temperature. Example 6 Synthesis of 3- (2-Bromo-4 (guanidinomethyl) phenoxy) propyl 4-bromobenzenesulfonate, hydrochloric salt or „or Λο ·

NH U

NH₂ HCI

Compound 4 (2.00 g, 2.77 mmol) was introduced into a 25 mL round bottom flask followed by HC1 (28.0 immoles; 7.00 mL of a 4.0 M solution in dioxane), and the resulting solution was stirred 4 h. The white solid obtained in this way was separated, washed thoroughly with MTBE (20 mL) and then dried to obtain a constant weight (1.4 g, 2.51 mmol; 90.6%). NMR (400 MHz, D₂O + DMSO-ds) δ 6.94 (d, 2H), 6.76 (d, 2H), 6.74 (s, 1H), 6.45 (m, 1H), 6.17 (d, 1H), 3.53 (m, 4H), 3.15 (t, 2H), 1.36 (m, 2H), 0.5 (s, 1H).

207

Example 7

Synthesis of 3- (2-Bromo-4 (guanidinomethyl) phenoxy) propyl 4-bromobenzenesulfonate, p-toluenesulfonic salt

B \.

<img file="MX367382B_D0093.tif" />

Compound 4 (0.50 g, 0.69 mmol), hydrated ptoluenesulfonic acid (1.32 g, 6.93 mmol) and THF (6 mL) were introduced into a 25 tnL round bottom flask. The resulting solution was heated to reflux under a nitrogen atmosphere, maintained 6 h and then slowly cooled to room temperature overnight. The white solid precipitate obtained in this way was separated, washed thoroughly with EtjO and dried to obtain a constant weight (0.328 g, 0.473 mmol; 68.3%).^TH NMR (300 MHz, DMSO-dg) δ 7.74 (m, 5H), 7.48 (d, 1H), 7.45 (m, 2H), 7.23 (dd, &lt;7 = 3 Hz, 1H), 7.08 (m, 3H ), 6.99 (d, &lt;7 = 9 Hz, 1H), 4.25 (m, &lt;7 = 6 Hz, 3H), 3.97 (t, &lt;7 = 6 Hz, 2H), 2.26 (s, 3H), 2.04 ( m, 2H).

Example 8

Synthesis of 3- (2-Bromo-4 (guanidinomethyl) phenoxy) propyl 4-bromobenzenesulfonate, acetic salt

208

The product of Example 6 (200 mg, 0.359 immoles) was dissolved in THF / H₂O (2 mL; 1: 1 v / v) was then treated with AgOAc (3 mL of a 22 mg / mL solution in 1: 4 MeCN / H₂O); immediate precipitation was observed. The dense suspension was stirred for 20 min, then filtered through a 0.45 pm PVDF filter disc and the filtrate was lyophilized. The amorphous salt obtained in this way was dissolved in CH2CI2 (1 mL), stirred 2 h at room temperature, and then cooled to 5 'C and kept at this temperature 3 h. The resulting white crystalline solids were filtered off and then air dried (0.100 g, 0.172 mmol; 48.0%).^XH NMR (400 MHz, DMSO-d_s) δ 9.6 (sa, 1H), 7.77 (m, 4H), 7.49 (d, 1H), 7.2 (m, 1H), 7.0 (d, 1H), 4.26 (m, 4H), 3.97 (t, 2H) , 2.06 (m, 2H), 1.66 (s, 3H). Example 9 Synthesis of 3- (2-Bromo-4 (guanidinomethyl) phenoxy) propyl 4-bromobenzenesulfonate, βγει benzoic salt product of Example 6 (415 mg, 0.744 mmol) was dissolved in THF / H₂O (4.2 mL; 1: 1 v / v) was then treated with AgOBz (10 mL of a 16 mg / mL solution in 1: 4 MeCN / H₂0); immediate precipitation was observed. The dense suspension was stirred for 20 min, then filtered through

209 a 0.45 pm PVDF filter disc and the filtrate was lyophilized. The amorphous salt obtained in this way was dissolved in EtOAc (10 mL), stirred 2 h at room temperature, and then cooled to 5 ° C and maintained at this temperature 3 h. The resulting white crystalline solids were filtered off, washed with EtOAc (1 mL) and then air dried (0.090 g, 0.140 mmol; 18.8%). NMR (400 MHz, DMSO-dg) δ 9.27 (sa, 1H), 7.88 (sa, 3H), 7.76 (m, 4H), 7.52 (d, 2H), 7.31 (m, 4H), 7.0 (d, 1H ), 4.26 (m, 4H), 3.97 (m, 2H), 2.06 (tn, 2H), 1.66 (s, 3H).

Example 10

Synthesis of 3- (2-Bromo-4 (guanidinomethyl) phenoxy) propyl 4-bromobenzenesulfonate, phosphoric salt

NH

<img file="MX367382B_D0094.tif" />

Compound 4 (0.200 g, 0.277 mmol) was dissolved in CH₂C1₂/ TFA (2 mL, 4: 1 v / v) and then stirred overnight at room temperature. Then, all volatiles were removed in vacuo and the resulting thick oil was subsequently dried in a vacuum oven (2 h at 25 ° C and Smbares). Subsequently, EtOAc (2 mL) and phosphoric acid (0.30 mmol; 62 pL of 5 M solution in THF) were added, and the resulting mixture was heated to reflux 3-5 min. After cooling to room temperature, MTBE (1 mL) was added. The

210 The resulting suspension was filtered through a sintered glass funnel, air dried and placed in a vacuum oven (48 hours at 25 'C and 5 pairs; 0.164 g, 2.65 mmol; 96.2%). ^XH NMR (400 MHz, D₂0 + DMSO-dJ δ 7.92 (c, 4H), 7.55 (s, 1H), 7.35 (m, 1H), 7.05 (d, 1H), 4.33 (m, 4H), 4.03 (t, 2H), 2.13 (m , 2H), 1.25 (s, 1.5H).

Example 11

Synthesis of 3 - (2-Bromo-4 (guanidinomethyl) phenoxy) propyl 4-bromobenzenesulfonate, methanesulfonic salt

Br ·

NH

OR

NH₂

Compound 4 (1.00 g, 1.38 mmol) was dissolved in CH₂C1₂ (8 mL), then treated with distilled TFA (2 mL) dropwise at room temperature and stirred overnight. All volatiles were removed, and the residue was treated successively with EtOAc (10 mL ·) and MsOH (1.52 mmol, 153 gL of a 10 M solution in THF). The resulting solution was heated to reflux, kept at this temperature 3-5 min and then slowly cooled to room temperature in the oil bath. The solid product was isolated by filtration, dried with air and then placed in a vacuum oven (48 h at 25 'C and Smbares; 0.838 g, 1.36 mmol; 98.6%). ^TH NMR (400 MHz, D₂O + DMSO-d_s) δ 7.7b (d, 4H), 7.5 (s, 1H), 7.26 (d,

211

1Η), 7.0 (d, 1H), 4.31 (m, 4H), 3.95 (t, 2H), 2.33 (s, 3H), 2.07 (m, 2H).

Example 12

Synthesis of 3- (2-Bromo-4 (guanidinomethyl) phenoxy) propyl 4-bromobenzenesulfonate, sulfuric salt

Εγει Compound 4 (0.100 g, 0.157 mmol) was suspended in dioxane (0.5 mL) then treated with sulfuric acid (0.158 mmol; 158 gL of a 1 M solution in THF) at room temperature; more dioxane (400 μύ) was needed to dissolve completely. The resulting solution was stirred several minutes and then concentrated in vacuo (overnight at 25 ° C and 5 umbrellas). The crude solid mass was washed with hot EtOAc, sonicated briefly and cooled before filtering. The resulting solid material was subsequently dried under vacuum to obtain the final product. ¹H NMR (400 MHz, D₂O + DMSO-d_and) 6 9.8 (s, 1H), 8.1 (s, 1H), 7.75 (q, 4H), 7.5 (d, 1H), 7.25 (saym, 4H), 7.0 (d, 1H), 4.25 (m, 4H) , 3.95 (t, 2H), 2.0 (m, 2H). Example 13 Synthesis of 3- (2-Bromo-4 (guanidinomethyl) phenoxy) propyl 4-methylbenzenesulfonate, trifluoroacetic salt (salt of

212

TFA of the tracer agent precursor-2)

<img file="MX367382B_D0095.tif" />

<img file="MX367382B_D0096.tif" />

Example 13A

Synthesis of 3- (4 - ((2,3-bis (tertbutoxycarboni1) guanidino) met i1) -2-bromophenoxy) propyl 4-methylbenzenesulfonate

NBoc

BrN NHBoc

H

Compound 3 (2.00 g, 3.98 mmol), 4-toluenesulfonyl chloride (0.987 g, 5.17 mmol), etc. were successively introduced into a round bottom flask.₃N (0.604 g, 5.97 mmol), DMAP (0.139 g, 1.19 mmol) and CH₂C1₂ (16 mL) at room temperature. After 5 h, the reaction mixture was poured into a separatory funnel, washed with water (10 mL) and brine (10 mL), and then dried with MgSOs, filtered and concentrated to obtain a foam. The solid was redissolved in CH₂C1₂(4 mL) was then introduced into a 40 g silica column (Redisep R_F) and was purified using a Teledyne ISCO Combiflash instrument (A: hexanes; B: EtOAc; 0-100% B in 19.2 min; 40 mL / min) to obtain the product as a white solid (1.89 g, 72.3%) . ³Η NMR (300 MHz, CDC1₃) δ 11.54 (s, 1H), 8.56 (C a, 1H), 7.75 (d, 2H, J = 4.5 Hz),

213

7.47 (d, 1H, J = 3 Hz), 7.22 (m, 3H), 6.75 (d, 1H, J = 4.5 Hz), 4.55 (d, 2H, J = 6 Hz), 4.32 (t, 2H, J = 6 Hz), 3.98 (t, 2H, J = 6 Hz), 2.36 (s, 3H), 2.16 (m, 2H), 1.54 (s, 9H), 1.50 (s, 9H).

Example 13B

Synthesis of 3- (2-Bromo-4 (guanidinomethyl) phenoxy) propyl 4-bromobenzenesulfonate, trifluoroacetic salt (TFA salt of the tracer-2 precursor)

o., or

Br ^

I nh o

AA

NH₂ F₃C OH H

In a round bottom flask the product of Example 13A (1.50 g, 2.28 mmol) was introduced and then treated with a solution of TFA in CH₂C1₂ at room temperature (52 mmol: 1: 1 v / v, 8 mL). After 3.5 h, the mixture was concentrated to obtain a thick oil, and then treated with acetone (2 mL) and concentrated again. The evaporation process with acetone was repeated two more times and the residue thus obtained was dissolved in CH₂C1₂ (4 mL). The CH₂C1₂ it was removed again in vacuo and the process was repeated twice more to obtain the crude product as a pale yellow solid. Finally, the solid was washed with MTBE (2 x 10 mL) and EtOAc (5 mL) to obtain the TFA salt of the tracer-2 precursor as a white, non-agglomerated powder.^ΧΗ NMR (300 MHz, DMSO-d₆) δ 7.98 (t, 1H,

214

<td>Hz), 7.74</td><td>(d, 2H, J</td><td colspan="2">= 9 Hz), 7.51 (d,</td><td>1H, J = 3</td><td>Hz), 7.34 (d,</td>

<td>2H, J = 9</td><td>Hz), 7.26</td><td colspan="2">(dd, 1H, J = 3, 9</td><td>Hz), 7.02</td><td>(d, 1H, J = 9</td>

<td>Hz), 4.30</td><td>(d, 2H, J</td><td>= 6 Hz),</td><td>4.22 (t,</td><td>2H, J = 6</td><td>Hz), 3.99 (t,</td>

<td>2H, J = 6</td><td>Hz), 2.35</td><td>(s, 3H),</td><td>2.07 (m,</td><td>2H).</td><td></td>

Example 14

Salt stability study

Long-term chemical integrity of several salt forms of the tracer-1 precursor was evaluated by monitoring the purity in percent by weight of solid samples at rest under controlled storage conditions: 40 and 70 C, and 60% relative humidity. The data shown in Figure 7 and that are tabulated in Table 1 list some of the differences observed. Example 15 Physical properties of selected salt forms

Next, selected physical properties of the salts of Examples 5-6 and 8-12 are tabulated, determined using established characterization methods (Table 1).

215

Table 1: Summary of the physical properties of salt forms

<td>t (d</td><td></td><td> 0</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> 0</td><td></td><td>c</td><td></td><td></td><td></td><td>Ό</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>k 0</td><td></td><td>• d</td><td> &gt;</td><td></td><td></td><td>&lt;0 • Ü</td><td></td><td>Φ</td><td></td><td></td><td>Q</td><td>OR</td><td></td><td></td><td></td>

<td></td><td>4J</td><td>&lt;d X »</td><td rowspan="2">g. Φ</td><td>«J k</td><td> 0</td><td> □</td><td></td><td>n</td><td></td><td>0) c</td><td> 4-&gt;</td><td></td><td></td><td></td><td></td>

<td>Ml • d</td><td>&lt;d</td><td>(/) • d</td><td>0) Cn</td><td>Η in</td><td>• d Λ λ</td><td></td><td></td><td></td><td>AND</td><td> 0)</td><td></td><td></td><td>Id</td><td></td>

<td>k</td><td>or</td><td>k OR</td><td>H</td><td>• d</td><td>Λ</td><td>L&gt; υ</td><td></td><td>ω</td><td></td><td>CU</td><td> 0)</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td> 0</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>you</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td> &gt;</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>0 N</td><td>4J in</td><td> &amp;</td><td></td><td>I</td><td></td><td></td><td>I</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>to</td><td>• d</td><td>Φ</td><td></td><td>rt</td><td></td><td></td><td></td><td></td><td></td><td>g</td><td></td><td></td><td>in</td><td></td>

<td></td><td>m</td><td>or</td><td>Rh</td><td></td><td></td><td></td><td></td><td>i</td><td></td><td></td><td> 1</td><td></td><td></td><td>H</td><td></td>

<td></td><td></td><td> 0</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>you</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>or</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>η</td><td>To U)</td><td> &amp;</td><td></td><td> &gt;</td><td></td><td></td><td>I</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>ω</td><td>♦ d</td><td>Φ</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>g</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>or</td><td>H</td><td></td><td> 1</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>or</td><td></td>

<td></td><td></td><td> 0</td><td></td><td></td><td></td><td></td><td>Φ</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>to</td><td></td><td></td><td></td><td></td><td>Ό</td><td></td><td></td><td></td><td>fd</td><td></td><td></td><td></td><td></td>

<td></td><td>0 AJ flj</td><td>d</td><td>&gt; • d</td><td></td><td>Ο ο Ο</td><td></td><td>the</td><td>CO</td><td></td><td>er</td><td> 0</td><td>uo</td><td></td><td></td><td></td>

<td></td><td> 44</td><td>« • d</td><td>σ Φ</td><td></td><td>k · Η σ »&amp;</td><td></td><td>υ N</td><td> 0) (0</td><td></td><td>AND</td><td>you</td><td>or</td><td></td><td>g</td><td></td>

<td></td><td>you in</td><td>k O</td><td>H</td><td></td><td>d Ό Μ Ο</td><td></td><td></td><td></td><td></td><td>CL</td><td>Φ</td><td></td><td></td><td></td><td></td>

<td></td><td></td><td> 0</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>you</td><td></td><td></td><td></td><td>n &lt;</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td> 0</td><td>rd</td><td></td><td></td><td></td><td></td><td></td><td> 0)</td><td></td><td></td><td>Q</td><td>or</td><td></td><td></td><td></td>

<td></td><td>(d</td><td>nJ u</td><td> &amp;</td><td>(okay</td><td>Ο</td><td>Fl o</td><td></td><td>X)</td><td></td><td>aj g</td><td>4J you</td><td></td><td></td><td></td><td></td>

<td></td><td>m</td><td>(0 • d</td><td>Φ</td><td>Φ Cn</td><td>tn</td><td> 8·</td><td></td><td></td><td></td><td>• d</td><td>0) í&gt;</td><td>in H</td><td></td><td></td><td></td>

<td></td><td>0 Pu</td><td>ku</td><td>H</td><td>• rt</td><td>Λ</td><td>0 υ</td><td></td><td>w</td><td></td><td>CU</td><td>Φ</td><td></td><td></td><td>or</td><td></td>

<td></td><td></td><td> 0</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td> 0</td><td>you • d</td><td></td><td></td><td></td><td>V Π5</td><td></td><td></td><td></td><td></td><td> &lt;0</td><td>r)</td><td></td><td></td><td></td>

<td></td><td>td</td><td>you</td><td>• d ti</td><td>ra</td><td>ω ο</td><td>U • d O</td><td></td><td>TO</td><td></td><td>er</td><td> 0</td><td>or</td><td></td><td></td><td></td>

<td></td><td>• r | W</td><td>V) • d</td><td>&amp; Φ</td><td>_tge</td><td>k tn</td><td> &amp;</td><td></td><td>you 4J</td><td></td><td>I υ</td><td>you 0) s</td><td>L52</td><td></td><td>co OI</td><td></td>

<td></td><td>s</td><td>k υ</td><td>H</td><td>kq</td><td> 5</td><td>0 u</td><td></td><td>M</td><td></td><td>CU</td><td>&lt;U</td><td></td><td></td><td>H</td><td></td>

<td></td><td> 0</td><td></td><td></td><td>nj</td><td></td><td>w</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>&lt;d</td><td>you</td><td></td><td></td><td>you</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>Ό</td><td></td><td></td><td></td><td></td><td>&lt;uk</td><td></td><td>Φ</td><td></td><td>i.</td><td>Λ</td><td></td><td></td><td></td><td></td>

<td></td><td>Λ</td><td>AJ</td><td> &amp;</td><td>υ</td><td>&lt;d</td><td>• dr ·</td><td></td><td>Λ</td><td></td><td>Φ g</td><td>AJ</td><td> 0</td><td></td><td></td><td></td>

<td></td><td> 0</td><td>• d</td><td>d)</td><td>τ)</td><td>k</td><td>A &lt;</td><td></td><td></td><td></td><td>♦ r4</td><td>0) s</td><td></td><td></td><td>g</td><td></td>

<td></td><td>or</td><td>M υ</td><td>r4</td><td>Μ Η</td><td>w Ul</td><td>OR/ Ό</td><td></td><td>ω</td><td></td><td>Oj</td><td> 0)</td><td>H</td><td></td><td> 1</td><td></td>

<td></td><td></td><td></td><td></td><td></td><td>Ό</td><td></td><td></td><td> &gt;.</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td>τί</td><td>Ή k</td><td></td><td> *3</td><td></td><td></td><td>ω</td><td></td><td></td><td></td><td></td><td></td><td> 0</td><td></td>

<td></td><td></td><td>* d</td><td>iet</td><td></td><td>or</td><td></td><td>the D</td><td>Ü&gt;</td><td>from</td><td></td><td>Tj</td><td></td><td>Ό nJ</td><td>k</td><td></td>

<td></td><td></td><td>you</td><td>ion</td><td></td><td>M4 OR OR</td><td></td><td>• H</td><td>m</td><td>YOU</td><td></td><td>• d</td><td> (0</td><td>• H</td><td></td><td></td>

<td></td><td></td><td>fd</td><td>to</td><td></td><td>OR)</td><td></td><td>• d</td><td> 0)</td><td>r ~</td><td></td><td>• d</td><td>OR</td><td></td><td> 0</td><td></td>

<td></td><td></td><td>u ü)</td><td>ϋβς</td><td></td><td>0 k ΟΊ</td><td></td><td>tai</td><td>c &lt;υ</td><td>or</td><td></td><td>XI Π5 x)</td><td>s</td><td>rd</td><td>0) υ</td><td>AND</td>

<td></td><td></td><td>k U</td><td>IT IS</td><td></td><td>• rl</td><td></td><td>It is</td><td>k 44</td><td>or</td><td></td><td>tn K</td><td>x!</td><td>SW</td><td></td><td>tn E</td>

216

The following examples (16-20) describe in detail the development of the combination of steps used for the manufacture of the tracer-1 agent. A flowchart of the overall process is shown in Figure 2. Example 16 Preparation of fluoride [¹⁸F]

Fluoride [¹⁸F] was obtained by proton bombardment of [^X8O] H2O in a cyclotron; The nuclear chemical transformation is shown below and can be summarized as¹⁸O (p, n)¹⁸F. For the purpose of bombing, the chemical form of ¹⁸Or is H2¹⁸O. The chemical form of ¹⁸The resulting F is the fluoride ion. ¹⁸0 + proton -> ¹⁸F + neutron

According to the procedures established in the industry, the [¹⁸O] H₂OR (2-3 mL) contained in a tantalum target body using Havar® foil was bombarded with 11 MeV protons (nominal energy); where the proton threshold energy for the reaction is 2.57 MeV and the maximum cross section energy is 5 MeV. The target volume, bombardment time and energy of protons can each be adjusted to control the amount of fluoride [¹⁸F] produced. Example 17

Synthesis of 1- {3-bromo-4- [3- [¹⁸F] f luoropropoxy] benzyljguanidine

217 (Tracer Agent-1)

Br ¹⁸F

<img file="MX367382B_D0097.tif" />

NH

OR

NH₂

H

The product of Example 16 was transferred from the cyclotron to the synthesis module and then filtered through an anion exchange column to remove the [¹⁸O] H2O that had not reacted; fluoride [¹⁸F] was retained in the cationic resin matrix. Then the column was washed with k2CO₃ aqueous and transferred to the reaction vessel. The resulting solution was diluted with MeCN, and then concentrated to dryness using an elevated temperature and reduced pressure. The [¹⁸Anhydrous F] KF thus obtained was treated individually with MeCN solutions of the products of Example 5E, 7 or 11 and Kryptofix 222, and then heated to 110 ° C and kept at this temperature for 15 min.

Example 17A

Synthesis of 1- {3-bromo-4- [3 [¹⁸F] fluoropropoxy] benayl Jguanidine, formic salt (formic acid salt of tracer-1)

Br ·

NH O

BA

NH₂ HiDH

H

218

Example 17B

Development of the preparative HPLC purification method

The selection of suitable parameters to purify the product of Example 17 was achieved by a detailed study of the chromatographic behavior of the product of several salts of the tracer-1 precursor. Initial column selection was carried out using a gradient of 9.5% / min from 5-95% MeCN containing 0.1% HCO₂H and 10% H₂Or with a flow of 1.00 mL / min, which showed a better specificity against known impurities when using the Agilent Zorbax BONUS-RP column (4.6 x 150 mm); Selected chromatograms are provided in Figures 8A-8B.

After selecting the column, a detailed study on the optimal solvent modifier was carried out, in which the counterion, concentration and ionic strength were adjusted to reach an ideal compromise between resolution and retention of the compound. Below is a table with a summary of the experimental parameters evaluated (Table 2).

219

Table 2: Summary of HPLC purification - TFA salt of tracer-1 precursor using Zorbax BONUS-RP from

Agilent

<td>Modifier</td><td>Concentration (mM)</td><td>pH</td><td>Strength ionic</td><td>Retention (min)</td><td>Tail factor</td>

<td>hco₂h</td><td> 22</td><td> 2.81</td><td></td><td> 6.88</td><td> 0.91</td>

<td>hco₂nh₄</td><td> 10</td><td> 3.13</td><td> 0.001</td><td> 6.74</td><td> 0.89</td>

<td>hco₂nh₄</td><td> 10</td><td> 3.97</td><td> 0.006</td><td> 7.45</td><td> 1.08</td>

<td>HCOjNH₄</td><td> 10</td><td> 4.5</td><td> 0.008</td><td> 7.71</td><td> 1.14</td>

<td>hco₂nh₄</td><td> 5</td><td> 4.5</td><td> 0.004</td><td> 7.76</td><td> 1.18</td>

<td>hco₂nh₄</td><td> 15</td><td> 4.5</td><td> 0.012</td><td> 7.91</td><td> 1.24</td>

<td>MeCOjNH,</td><td> 10</td><td> 4.03</td><td> 0.001</td><td> 6.77</td><td> 0.82</td>

<td>MeCOjNHí</td><td> 10</td><td> 4.46</td><td> 0.003</td><td> 7.34</td><td> 1.04</td>

<td>Dumb₂NH,</td><td> 10</td><td> 5.48</td><td> 0.008</td><td> 8.01</td><td> 1.32</td>

Example 17C

Synthesis of 1- {3-bromo-4- [3 [¹⁸F] fluoropropoxy] benzyljguanidine, formic salt

<img file="MX367382B_D0098.tif" />

The product of Example 17 was cooled to room temperature and the solution was concentrated. The crude product was diluted with H₂0 / MeCN (1 mL, 4: 1 v / v), then purified directly by HPLC on a Zorbax column

Agilent BONUS-RP using an NH solution₄HCO₂ in

220

H₂O / MeCN. The peak of the main product was collected and analyzed for purity and radiochemical performance.

Table 3: Summary of the purity and radiochemical performance of various salt forms of the precursor

<td></td><td>Precursor of tracer agent-1</td><td>Ex emplo 7</td><td>Example 11</td>

<td>Radiochemical performance Radiochemical purity</td><td> 60% 99%</td><td> 35% 100%</td><td> 15% 99%</td>

Example 18

General preparation of tracer agent-1

The following example describes a general procedure for synthesizing the tracer-1 agent using an automated synthesis module. Fluoride [¹⁸Aqueous F] prepared in Example 16 was transferred from the cyclotron to a synthesis module and then filtered through an anion exchange column to remove the [¹⁸O] H₂Or that he had not reacted; fluoride [¹⁸F] was retained in the cationic resin matrix. Then, the column was washed with an aqueous base and transferred to the reaction vessel. The resulting solution was optionally diluted with MeCN, and then concentrated to dryness using an elevated temperature and reduced pressure. The mixture of anhydrous fluoride [F] and base obtained in this way was treated with a

221 solution of the tracer-1 precursor (or a salt thereof), optionally an activating agent, and then heated to 90-110 ° C and maintained at this temperature 5-15 min. After cooling, the solution was evaporated to dryness using an elevated temperature and reduced pressure, and then reconstituted in H₂O / MeCN and purified directly by HPLC on an Agilent BONUS-RP column using an NH solution₄HC0₂ in H₂O / MeCN. The peak of the main product was collected, diluted with ascorbic acid and then transferred to the formulation module.

Example 18A-1

Preparation of the formic salt of tracer-1 agent using the Modular-Lab synthesis module of Eckert &amp; Ziegler

The product of Example 15 was transferred from a cyclotron to the synthesis module and then filtered through an anion exchange column to remove the [¹⁸O] H2O that had not reacted; fluoride [¹⁸F] was retained in the cationic resin matrix. Then the column was washed with K2CO₃ (11.5 pmol; 0.500 mL of a 23.0 mM solution in H₂O) and transferred to the reaction vessel. The resulting solution was diluted with MeCN (0.500 mL) and then concentrated to dryness using a two step procedure; heating up to 135 'C for 5 min under vacuum and with nitrogen flow (500 mL / min), already

222 then at 100 'C for 10 tnin under vacuum and with nitrogen flow (500 mL / min). The mixture of [^ieF] KF anhydrous and K₂CO₃obtained in this way was treated with a solution of the TFA salt of the tracer-1 precursor (5.00 mg, 7.87 umol) K and Kryptofix 222 (22.5 mg, 59.7 pmol) in t-BuOH: MeCN (4: 1 v / v; 1.5 mL), and then heated to 110 ° C and maintained at this temperature 15 min. The resulting solution was cooled to 95 ° C and then concentrated for 5 min with a stream of nitrogen. Subsequently, the mixture was treated with H₂O / MeCN (4: 1 v / v; 1.00 mL) and heated to 100 ° C for 5 min. After cooling for 60 s, the resulting solution was purified directly by HPLC on an Agilent BONUS-RP column (10 pm; 9.4 x 250 mm) using an eluent 82:18 of H₂O / MeCN containing NH₄HCO₂ (pH 3.8) with a flow of 5 mL / min. The peak of the eluted main product was collected at 12-14 min, diluted with ascorbic acid (10 mL of a 0.28 M solution in H₂OR; pH 4) and then transferred to the formulation module; 50% radiochemical performance corrected for decay.

Example 18A-2

Preparation of the formic salt of tracer-1 agent using the Modular-Lab synthesis module of Eckert &amp; Ziegler

The product of Example 16 was transferred from the cyclotron to the synthesis module and then filtered through

223 an anion exchange column to remove the [¹⁸O] H2O that had not reacted; fluoride [¹⁸F] was retained in the cationic resin matrix. Then the column was washed with K2CO₃ (2.01 pmol; 0.500 mL of a 4.02 mM solution in H₂O) and transferred to the reaction vessel. The resulting solution was diluted with MeCN (0.500 mL) and then concentrated to dryness using a two step procedure; heating up to 135 'C for 3 min under vacuum and with nitrogen flow (500 mL / min), and then at 100' C for 9 min under vacuum and with nitrogen flow (500 mL / min). The mixture of [¹⁸F] KF anhydrous and K₂CO₃obtained in this way was treated with a solution of the product of Example 7 (1.00 mg, 1.44 μιηοΐ) and Kryptofix® 222 (4.11 mg, 11.0 μπιοί) in t-BuOH: MeCN (4: 1 v / v; 1.5 mL), and then heated to 110 'C and kept at this temperature 15 min. The resulting solution was cooled to 95 ° C and then concentrated for 5 min with a stream of nitrogen. Subsequently, the mixture was treated with H₂O / MeCN (4: 1 v / v; 1.00 mL) and heated to 100 ° C for 5 min. After cooling for 60 s, the resulting solution was purified directly by HPLC on an Agilent BONUS-RP column (10 μιη; 9.4 x 250 mm) using an 82:18 eluent of H₂0 / MeCN containing NH₄HCO₂ (pH 3.8) with a flow of 5 mL / min. The peak of the eluted main product was collected at 12-14 min, diluted with ascorbic acid (10 mL of a 0.28 M solution in

224

H₂OR; pH 4) and then transferred to the formulation module; 33% radiochemical performance corrected for decay.

Example 18A-3

Preparation of the formic salt of tracer-1 agent using the Modular-Lab synthesis module of Eckert &amp; Ziegler

The product of Example 16 was transferred from the cyclotron to the synthesis module and then filtered through an anion exchange column to remove the [¹⁸O] H2O that had not reacted; fluoride [¹⁸F] was retained in the cationic resin matrix. Then the column was washed with K2CO₃ (2.01 pmol; 0.500 mL of a 4.02 mM solution in H₂0) and transferred to the reaction vessel. The resulting solution was diluted with MeCN (0.500 mL) and then concentrated to dryness using a two step procedure; heating up to 135 'C for 3 min under vacuum and with nitrogen flow (500 mL / min), and then at 100' C for 9 min under vacuum and with nitrogen flow (500 mL / min). The mixture of [^ieF] KF anhydrous and K₂CO₃obtained in this way was treated with a solution of the product of Example 11 (0.88 mg, 1.44 prnol) and Kryptofix® 222 (4.11 mg, 11.0 pmol) in t-BuOH: MeCN (4: 1 v / v; 1.5 mL), and then heated to 110 ° C for 15 min. The resulting solution was cooled to 95 ° C and then

225 concentrated for 5 min with a stream of nitrogen. Subsequently, the mixture was treated with H₂O / MeCN (4: 1 v / v; 1.00 mL) and heated to 100 ° C for 5 min. After cooling for 60 s, the resulting solution was purified directly by HPLC on an Agilent BONUS-RP column (10 μπί; 9.4 x 250 mm) using an eluent 82:18 of H₂0 / MeCN containing NH₄HCO₂ (pH 3.8) with a flow of 5 mL / min. The peak of the eluted main product was collected at 12-14 min, diluted with ascorbic acid (10 mL of a 0.28 M solution in H₂OR; pH 4) and then transferred to the formulation module; 15% radiochemical performance corrected by decay. Example 18A-4 Preparation of the formic salt of tracer agent-1 using the Modular-Lab synthesis module of Eckert &amp; Ziegler

The product of Example 16 was transferred from the cyclotron to the synthesis module and then filtered through an anion exchange column to remove the [¹⁸O] H2O that had not reacted; fluoride [^ieF] was retained in the matrix of the aationic resin. Then the column was washed with Et4NHCO₃ (39.4 pmol; 0.500 mL of a 78.8 mM solution in H₂O) and transferred to the reaction vessel. The resulting solution was diluted with MeCN (0.500 mL) and then concentrated to dryness using a

226 two stage procedure; heating up to 135 'C for 5 min under vacuum and with nitrogen flow (500 mL / min), and then at 100' C for 10 min under vacuum and with nitrogen flow (500 mL / min). The mixture of [¹⁸F] Et₄NF anhydrous and Et₄NHCO₃ obtained in this way was treated with a solution of the tracer-1 precursor (5.00 mg, 7.87 pmol) in tBuOH: MeCN (4: 1 v / v; 1.0 mL), and then heated to 110 ° C for 15 min . The resulting solution was cooled to 95 ° C and then concentrated for 5 min with a stream of nitrogen. Subsequently, the mixture was treated with H₂O / MeCN (4: 1 v / v; 1.00 mL) and heated to 100 ° C for 5 min. After cooling for 60 s, the resulting solution was purified directly by HPLC on an Agilent BONUS-RP column (10 gm; 9.4 x 250 mm) using an eluent 82:18 of H₂O / MeCN containing NH₄HCO₂ (pH 3.8) with a flow of 5 mL / min. The peak of the eluted main product was collected at 12-14 min, diluted with ascorbic acid (10 mL of a 0.28 M solution in H₂0; pH 4) and then transferred to the formulation module; 46% radiochemical performance corrected by decay.

Example 18A-5

Preparation of the formic salt of tracer-1 agent using the Modular-Lab synthesis module of Eckert &amp; Ziegler

The product of Example 16 was transferred from the cyclotron

227 to the synthesis module and then filtered through an anion exchange column to remove the [¹⁸O] H2O that had not reacted; fluoride [^ieF] was retained in the cationic resin matrix. Then the column was washed with Et4NHCO₃ (31.5 pmol; 0.500 mL of a 63.0 mM solution in H₂0) and transferred to the reaction vessel. The resulting solution was diluted with MeCN (0.500 mL) and then concentrated to dryness using a two-stage procedure; heating up to 13 5 'C for 5 min under vacuum and with nitrogen flow (500 mL / min), and then at 100' C for 10 min under vacuum and with nitrogen flow (500 mL / min). The mixture of [^youF] Et₄NF anhydrous and Et₄NHCO₃ obtained in this way, it was treated with a solution of the TFA salt of the tracer-1 precursor (4.00 mg, 6.30 pmol) in MeCN (1.0 mL), and then heated to 110 ° C for 15 min. The resulting solution was cooled to 95 ° C and then concentrated for 5 min with a stream of nitrogen. Subsequently, the mixture was treated with H₂O / MeCN (4: 1 v / v; 1.00 mL) and heated to 100 ° C for 5 min. After cooling for 60 s, the resulting solution was purified directly by HPLC on an Agilent BONUS-RP column (10 μαΐ; 9.4 x 250 mm) using an eluent 82:18 of H₂O / MeCN containing NH₄HCO₂ (pH 3.8) with a flow of 5 mL / min. The peak of the eluted main product was collected at 12-14 min, diluted with ascorbic acid (10 mL of a 0.28 M solution in

228

H₂0; pH 4) and then transferred to the formulation module; 42% radiochemical performance corrected for decay.

Example 18A-6

Preparation of the formic salt of tracer-1 agent using the Modular-Lab synthesis module of Eckert &amp; Ziegler

The product of Example 16 was transferred from the cyclotron to the synthesis module and then filtered through an anion exchange column to remove the [¹⁸0] H20 that had not reacted; fluoride [¹⁸F] was retained in the cationic resin matrix. Then the column was washed with Et4NHCO₃ (39.5 pmol; 0.500 mL of a 79.0 mM solution in H₂0) and. It was transferred to the reaction vessel. The resulting solution was diluted with MeCN (0.500 mL) and then concentrated to dryness using a two step procedure; heating up to 135 'C for 5 min under vacuum and with nitrogen flow (500 mL / min), and then at 100' C for 10 min under vacuum and with nitrogen flow (500 mL / min). The mixture of [^1SF] Et₄NF anhydrous and Et₄NHCO₃ obtained in this way was treated with a solution of the TFA salt of the tracer-2 precursor (4.50 mg, 7.87 pmol) in MeCN (1.0 mL), and then heated to 110 ° C and kept at this temperature for 15 min. The resulting solution was cooled to 95 'C and then

229 concentrated for 5 min with a stream of nitrogen. Subsequently, the mixture was treated with H₂0 / MeCN (4: 1 v / v; 1.00 mL), heated to 100 ° C and kept at this temperature 5 min. After cooling 60 s, the resulting solution was purified directly by HPLC on an Agilent BONUS-RP column (10 gm; 9.4 x 250 mm) using an eluent 82:18 of H_z0 / MeCN containing NH₄HCO₂ (pH 3.8) with a flow of 5 mL / min. The peak of the eluted main product was collected at 12-14 min, diluted with ascorbic acid (10 mL of a 0.28 M solution in H₂0; pH 4) and then transferred to the formulation module; 46% radiochemical performance corrected by decay. Example 18B-1 Preparation of the formic salt of tracer-1 agent using GE's TRACERLab MX synthesis module

The product of Example 16 was transferred from the cyclotron to the synthesis module and then filtered through an anion exchange column to remove the [¹⁸O] H2O that had not reacted; fluoride [^iaF] was retained in the cationic resin matrix. Then the column was washed with K2CO₃ (11.5 pmol; 0.800 mL of a solution

14.4 mM in H₂O) and transferred to the reaction vessel. Subsequently, the resulting solution was concentrated to dryness using a two stage procedure; heating up to 95 C for 3 min under vacuum and with nitrogen flow,

230 and then at 115 'C for 7 min under vacuum and with nitrogen flow. The mixture of [¹⁸F] KF anhydrous and K₂CO₃ obtained in this way was treated with a solution of the TFA salt of the tracer-1 precursor (5.00 mg, 7.87 pmol) and

Kryptofix 222 (22.5 mg, 59.7 pmol) in t-BuOH: MeCN (4: 1 v / v;

1.5 mL), and then heated to 110 'C and kept at this temperature 15 min. The resulting solution was cooled to 95 ° C and then concentrated for 7 min with a stream of nitrogen. Subsequently, the mixture was treated with H₂O / MeCN (4: 1 v / v; 5.00 mL) and then heated to 95 ° C for 5 min. After cooling to 50 ° C, the resulting solution was purified directly by HPLC on an Agilent BONUS-RP column (10 μπί; 9.4 x 250 tran) using an 82:18 eluent of H₂O / MeCN containing NH₄HCO₂ (pH 3.8) with a flow of 5 mL / min. The peak of the eluted main product was collected at 10-12 min, diluted with ascorbic acid (10 mL of a 0.28 M solution in H₂0; pH 4) and then transferred to the formulation module; 20% radiochemical performance corrected for decay. In the

Figure 3 provides the flow chart for the process described above.

Example 18B-2

Preparation of the formic salt of the tracer-1 agent using the GE TRACERLab MX synthesis module

The product of Example 16 was transferred from the cyclotron

231 alο to the synthesis module and then filtered through an anion exchange column to remove the [¹⁸O] H2O that had not reacted; fluoride [¹⁸F] was retained in the cationic resin matrix. Then the column was washed with Et4NHCO₃ (39.5 pmol; 0.500 mL of a 79.0 mM solution in H₂0) and transferred to the reaction vessel. Subsequently, the resulting solution was concentrated to dryness using a two stage procedure; heating up to 95 'C for 3 min under vacuum and with nitrogen flow, and then at 115' C for 7 min under vacuum and with nitrogen flow. The mixture of [¹⁸F] Et₄NF anhydrous and Et₄NHCO₃ obtained in this way was treated with a solution of the TFA salt of the tracer-2 precursor (4.50 mg, 7.87 pmol) in MeCN (1.0 mL), and then heated to 90 ° C and kept at this temperature for 10 minutes. The resulting solution was cooled to 95 ° C and then concentrated for 7 min with a stream of nitrogen. Subsequently, the mixture was treated with H₂O / MeCN (4: 1 v / v; 2.00 mL), was heated to 90 'C and maintained at this temperature for 5 min. After cooling to 50 ° C, the resulting solution was purified directly by HPLC on an Agilent BONUS-RP column (10 μπί; 9.4 x 250 mm) using an 82:18 eluent of H₂O / MeCN containing NH₄HCO₂ (pH 3.8) with a flow of 5 mL / min. The peak of the eluted main product was collected at 10-12 min, diluted with ascorbic acid

232 (10 mL of a 0.28 M solution in H₂0; pH 4) and then transferred to the formulation module. The flowchart for the process described above is provided in Figure 4.

Example 18C

Preparation of the formic salt of the tracer-1 agent using the GE TRACERLab FX synthesis module

The product of Example 16 was transferred from the cyclotron to the synthesis module and then filtered through an anion exchange column to remove the [¹⁸O] H2O that had not reacted; fluoride [¹⁸F] was retained in the cationic resin matrix. Then the column was washed with K2CO₃ (11.5 pmol; 0.800 ml> of a solution

14.4 tnM in H₂0) and transferred to the reaction vessel. Subsequently, the resulting solution was concentrated to dryness using a two stage procedure; heating up to 68 C for 3 min under vacuum and with helium flow, and then at 95 'C for 4 min under vacuum and with helium flow. The mixture of [¹⁸F] KF anhydrous and K₂CO₃ obtained in this way it was cooled to 70 ° C, treated with a solution of the TFA salt of the tracer agent precursor-1 (5.00 mg, 7.87 pmol) and Kryptofix® 222 (22.5 mg, 59.7 pmol) in t-BuOH ; MeCN (4: 1 v / v; 1.5 mL), and then heated to 95 ° C and maintained at this temperature 15 min. The resulting solution was cooled to 55 ° C and then concentrated for 7 min.

233 With a stream of helium. The mixture was subsequently treated with H₂0 (0.1 mL), kept under these conditions 2 min, then cooled to 40 ° C and diluted with H₂O / MeCN (4: 1 v / v; 3.00 mL). The resulting solution was purified directly by HPLC on an Agilent BONUS-RP column (10 μη; 9.4 x 250 mm) using an eluent 82:18 of H₂0 / MeCN containing NH₄HCO₂ (pH 3.8) with a flow of 5 mL / min. The peak of the eluted main product was collected at 9-11 min, diluted with ascorbic acid (10 mL of a 0.2 8 M solution in H₂OR; pH 4) and then transferred to the formulation module; 40% radiochemical performance corrected for decay.

Example 18D

Preparation of the formic salt of the tracer-1 agent using the Siemens Explore RN synthesis module

The product of Example 16 was transferred from the cyclotron to the synthesis module and then filtered through an anion exchange column to remove the [¹⁸O] H2O that had not reacted; fluoride [¹⁸F] was retained in the cationic resin matrix. Then the column was washed with K2CO₃ (11.5 pmol; 0.800 mL of a solution

14.4 mM in H₂0) and transferred to the reaction vessel. Subsequently, the resulting solution was concentrated to dryness using a two stage procedure; heating up to 95 'C for 2 min under vacuum and with nitrogen flow,

2. 3. 4 and then at 115 C for 5 rain under vacuum and with nitrogen flow. The mixture of [¹⁸F] Anhydrous KF and K2CO3 thus obtained were treated successively with a solution of the TFA salt of the tracer-1 precursor (4.00 mg, 6.30 pmol) in MeCN (1.00 mL) and Kryptofix⁰ 222 (18.0 mg, 47.8 pmol) also in MeCN (0.50 mL), and then heated to 110 ° C and maintained at this temperature for 15 min. The resulting solution was cooled to 95 ° C and then concentrated for 5 min with a stream of nitrogen. Subsequently, the mixture was cooled to 55 ° C, treated with H20 / MeCN (4: 1 v / v; 1.00 mL) and purified directly by HPLC on an Agilent BONUS-RP column (10 gm; 9.4 x 250 mm ) using an eluent 82:18 of H₂O / MeCN containing NH₄HC0₂ (pH 3.8) with a flow of 5 mL / min. The peak of the eluted main product was collected at 12-14 min, diluted with ascorbic acid (10 mL of a 0.2 8 M solution in H₂0; pH 4) and then transferred to the formulation module, - 32% radiochemical yield corrected by decay.

Example 18E

Preparation of the formic salt of tracer agent-1 using the Explora GN synthesis module

The product of Example 16 was transferred from the cyclotron to the synthesis module and then filtered through an anion exchange column to remove the [¹⁸O] H₂Or that he had not reacted; fluoride [¹⁸F] was held

235 in the cationic resin matrix. Then the column was washed with Et₄NHCO₃ (39.5 pmol; 1.00 mL of a 39.5 mM solution in H₂O) and transferred to the reaction vessel. The resulting solution was diluted with MeCN (1.00 mL) and then concentrated to dryness; 110-115 'C. Then, more MeCN (1.50 mL) was added and the solution was re-concentrated to dryness. The mixture of [^ieF] Et₄NF anhydrous and Et₄NHCO₃ obtained in this way was treated with a solution of the TFA salt of the tracer-2 precursor (4.50 mg, 7.87 pmol) in MeCN (1.0 mL), and then heated to 90 ° C and kept at this temperature for 10 minutes. The resulting solution was cooled to 60 ° C and then concentrated to dryness; 95 ° C Subsequently, the mixture was treated with H₂0 / MeCN (4: 1 v / v; 2.00 mL), heated to 100 ° C and kept at this temperature 5 min. After cooling to 60 'C, the resulting solution was purified directly by HPLC on an Agilent BONUS-RP column (10 pm; 9.4 x 250 mm) using an eluent 82:18 of H₂O / MeCN containing NH4HCO2 (pH 3.8) with a flow of 5 mL / min. The peak of the eluted main product was collected at 12-14 min, diluted with ascorbic acid (10 mL of a 0.28 M solution in H₂OR; pH 4) and then transferred to the formulation module. The flowchart for the process described above is provided in Figure 5.

236

Example 19

Solvent Exchange

Tracer-1 agent was transferred from the purification module to the formulation module and then filtered through a tC18 Sep-Pak cartridge to remove the MeCN; tracer-1 agent was retained in matrix C18 and the filtrate was discarded. The cartridge was washed successively with ascorbic acid (10 mL of a 0.28 M solution in H₂0; pH 4), the filtrate was discarded, and then with EtOH / H₂O (1.00 mL; 1: 1 v / v) and the filtrate was collected. The ethanol concentrate obtained in this way was subsequently diluted with ascorbic acid (9.0 mL of a 0.28 M solution in H₂0; pH 5.8) as a preparation for final aseptic filtration. Example 20 Aseptic filtration process

The final product vial assembly was constructed from the following previously sterilized components: a 30 mL product vial, a Millipore Millex GV4 purge filter (0.22 pm x 4 mm), a tuberculin syringe (1 mL) and an insulin syringe (0.5 mL). Next, the product of Example 19 was transferred from the formulation module to the assembly of the final product vial through a Millex GV PVDF sterilizing filter from Millipore (0.22 pm x 13 mm). Next, samples were taken for quality control, using the syringe assemblies, to complete all the requirements for the development of the

237 product.

Example 21

The evaluation of several experimental parameters in the nucleophilic fluorination of the tracer-1 precursor using the combination of reagents K₂C0₃/ Kryptof ίχ^Φ 222 initially indicated that although the complexity of the overall reaction increased with the addition of K₂CO₃, the efficacy of fluorination remained unchanged for values ​​greater than 0.66 molar equivalents (Figure 9A). High levels of base (e.g., carbonate) were fundamentally correlated with a fruitless consumption of the starting material (eg, the precursor of the tracer-1 agent), the hydrolysis resulting in the alcohol derived from the route of primary decomposition Various combinations of alternative bases (Table 4), including the modification of potassium counterion as well as the substitution of organic amino bases, proved to be less effective as promoters of the fluorination reaction (&lt;10% conversion). Table 4. Comparison between base identity and fluoridation performance

<td>Base</td><td>% from performance</td>

<td>K₂CO₃</td><td> 45-60</td>

<td>KHSO₄</td><td> &lt;10</td>

<td>K₂HPO₄</td><td> &lt;10</td>

238

<td>Kh₂PO₄</td><td> &lt;10</td>

<td>i-Pr₂NEt</td><td> &lt;10</td>

<td>Tetramethylguanidine</td><td> &lt;10</td>

<td>Pyridine</td><td> &lt;10</td>

A lower fluoridation performance was also observed in Φ absence of Kryptofix 222, regardless of the stoichiometry of K₂CO₃. However, the presence of Kryptofix 222 significantly increased the pH of the solution (10-12).

The fluorination reaction was also evaluated with various solvent systems, including MeCN, t-BuOH and mixtures thereof; DMF, DMSO and THF alone. The combinations of MeCN and tBuOHzMeCN proved to be the most effective. Analysis of crude reaction mixtures of each solvent combination provided a specific impurity profile resulting from the fruitless consumption of the tracer-1 precursor (Figure 9B). The MeCN only provided the best combination of fluoridation efficiency and overall impurity profile.

A series of subsequent studies indicated that both the

0 Liberation of ^1SF of the anion exchange column as fluoridation efficiency are greatly affected by the identity, concentration and composition of the basic solution used during the transfer of ^iaF from the cyclotron to the reaction vessel. Specifically, we have noted that regardless of the identity of the cation (e.g., potassium or tetraalkylammonium

239 such as tetraethylammonium or tetrabutylammonium), there is a threshold concentration of the ammonium solution component (H0 ', HCO₃', MsO', TsO ', I'), below which there was a reduction in the effectiveness of the release of ¹⁸F. However, it should be noted that the effective release of ¹⁸F was not necessarily associated with effective fluoridation. Within only the tetrabutylammonium series (Table 5), we have determined that although the bicarbonate anion was superior to other anions, the reaction efficiency was lower than with the combination of K₂C0₃/ Kryptof ix ° 222 described above (e.g., Tables 4-5). With the increase in bicarbonate concentration, the overall fluoridation efficiency was improved. Figure 9C shows the effect of tetraalkylammonium bicarbonate concentration used for anion exchange on fluoridation efficiency. The combination of five molar equivalents of Et₄NHCO₃ and of the precursor of tracer agent-1 or -2 provided identical fluorination efficiency as well as an improved overall impurity profile compared to the K system₂C0₃/ Kryptofίχ ^ 222 original.

Table 5. Comparison of tetrabutylammonium salt forms and fluorination yield.

T% of ”

Saline form performance

Mesylate &lt;2

240

<td>Hydroxide</td><td> &lt;5</td>

<td>Tosylate</td><td> &lt;10</td>

<td>I last</td><td> 18.7</td>

<td>Baking Soda (8.8 mM)</td><td> 28.0</td>

<td>Baking soda (34.7 mM)</td><td> 60.0</td>

The non-radioactive experiments described above were adapted for the manufacture of the tracer-1 agent both in the Siemens RN remote synthesis module and in the Eckert &amp; Ziegler ModularLab. Thus, in the individual modules, multiple variable selection studies (base, time and temperature) were carried out that provided the specific parameters for each unit necessary to maintain chemical fidelity in discrete instruments; Example 18 describes the specific parameters. Example 22

A human study was conducted that determined the quantification of the normal pattern of the regional myocardial readioactivity concentration of the tracer-1 agent.

Methods: -220 MBq of the tracer-1 agent was injected into normal subjects (n = 6) intravenously and dynamic images were acquired by PET in 80 min with the patient still.

241

The images corrected for attenuation were reoriented in specific standard cardiac axes and the maximum regional myocardial absorption was quantified by sectors using the WLCQ software. The hearts were divided into three short axial sections (Base-B; Med-M; Apical-A) and four radial sectors (Anterior-A; Septal-S; Lower-I; Lateral-L) and the mean regional absorption was calculated For each sector. The activity was expressed as Bq / tnL.

Results: The radiotracer was rapidly removed from the blood and demonstrated a favorable biodistribution for early cardiac tomography. Regional and global myocardial activity peaked in the first 10 min and stabilized ~ 60 min after injection. There was no significant variation (p = 0.69, ANOVA) in regional myocardial absorption at this time around the circumference of the heart (A: 11592 + 2474 Bq / mL; S: 11647 ± 2829 Bq / mL; I: 11818 + 1991 Bq / mL; L: 11424 + 2439 Bq / mL). Nor was there a significant gradient of the apex base (p = 0.08, ANOVA) in myocardial absorption (B: 11284 ± 2844 Bq / mL; M: 11898 + 2047 Bq / mL; A: 11678 + 2148 Bq / mL).

The concentration of myocardial radioactivity of the tracer-1 agent was uniform throughout the heart in normal volunteers. This study established the normal pattern of quantitative regional myocardial radioactivity concentration. This type of regional myocardial analysis

242 It provides advantages compared to the evaluation of the proportions of the heart versus the mediastinum in future studies of patients with heart disease.

Example 23

The dosimetry of the tracer-1 agent in nonhuman primates was examined. Tracer-1 agent, marked with¹⁸F, is a novel norepinephrine transporter (NET) ligand and was a useful radiotracer for mapping cardiac nerve endings in vivo using positron emission tomography. A study was conducted on four nonhuman primates to estimate the dosimetry of human radiation.

Methods: In this study, a tomography of two male and two female cynomologists was performed using a Concord Focus 220 MicroPET scanner to determine the distribution of ¹⁸F throughout the body after a single intravenous injection of 4-5 mCi (0.65-1.6 pg) of the tracer-1 agent. Images of animals anesthetized with isofluorane were acquired from the head to the lower abdomen in 5 segments within four and a half hours after injection. Radioactivity in identifiable organs and in the rest of the body was determined as a function of time using an analysis of the regions of interest. The total number of disintegrations per unit injected was determined by normalizing the

243 radioactivity injected and integrating time-related data for radioactivity Erente to time. Using the OLINDA / EXM software (Organ Leve! Internal! Dose Assessment / EXponential Modeling Software, published by Vanderbilt University), the standardized number of disintegrations of^ieF for each organ was combined with the energy released in each disintegration, and using the MIRD scheme, estimates were made on the fraction of the total energy released that was retained in each source organ and the contribution of each source organ to the energy deposited in surrounding target organs for an adult human being. By dividing the total fractional energy deposited in each organ by the mass of the corresponding organ, the radiation dose for the organ was obtained per unit of injected dose (mCi or MBq).

Results: From the estimates of radiation doses, it was predicted that the human organ that would receive the highest dose was the wall of the urinary bladder with a mean of 0.41 +0.089 rem / mCi. The next five organs with the highest doses and their respective mean dose estimates were the kidneys (0.15 ± 0.088 rem / mCi), adrenal glands (0.14 +0.027 rem / mCi), heart wall (0.085 +0.014 rem / mCi) , osteogenic cells (0.084 ± 0.0048 rem / mCi) and red bone marrow (0.083 ± 0.0099 rem / mCi). The average whole body dose estimate was

0.044 ±0.00031

244 retn / mCi, and the mean effective dose defined in ICRP 60 was 0.070 ± 0.0059 rem / mCi. Refer to Example 25 below for more information on the effective dose.

Based on the mean values, it was estimated that the maximum dose of tracer-1 agent that can be administered to a human being without exceeding 50 mSv (5 rem) in the urinary bladder was 12 mCi. Similarly, it was estimated that the maximum administered dose that does not exceed the effective dose of 10 mSv is 14 mCi. Example 24

The following example describes the biodistribution and dosimetry in the organs for the tracer-1 agent.

Biodistribution and overall dosimetry in the organs for the tracer-1 agent labeled with ¹⁸F were determined based on the data of the images obtained by PET of 12 healthy subjects. Image quantification, modeling of kinetics were carried out to determine residence times and a dosimetric analysis.

Image data were obtained from the head to the mid-thigh obtained by PET for twelve healthy subjects using the tracer-1 agent labeled with ^1SF approximately 17, 31, 45, 117, 190 and 225 minutes after injection. In addition, images of the legs were also obtained approximately 66 and 274 minutes after the injection. The image data was corrected by attenuation in the center where

245 performed the tomography and were quantified based on methodology 16 for medical internal radiation dosimetry (MIRD) to determine kinetic data in all organs that show a significant absorption of activity. Dosimetry estimates were created using kinetic models of quantified image data to determine residence times and standard MIRD methodology using a similar method. Kinetic data, residence times and dosimetry estimates for each individual are indicated, as well as a summary of the statistical data.

Results:

No adverse events due to tracer-1 were observed. Initially, approximately 1.6% of the injected dose (DI) in the myocardium was observed, with more than 1.5% of the DI (corrected for decay) remaining after 4 hours of injection. The proportion of radioactivity in the myocardium with respect to the liver was initially approximately one and increased to more than two at 4 hours. Blood radioactivity was rapidly eliminated and lung activity was low throughout the study. On average, the organ that presented the highest maximum absorption was the urinary bladder with approximately 18.3% of the injected activity. The next highest maximum absorption occurred in the kidneys with approximately one

246

15.5% of the injected activity.

Dosimetry estimates: On average, the organ that received the highest absorbed dose was the urinary bladder wall, with 0.38 rem / mCi (0.10 mSv / MBq), followed by the kidneys with 0.31 rem / mCi (0.083 mSv / MBq ). The mean SD (effective dose) was 0.096 rem / mCi (0.026 mSv / MBq). Table 9 shows a summary of the statistical data for the absorbed dose in rem / mCI for all subjects. Table 10 shows a summary of the statistical data for the absorbed dose in mGy / MBq for all subjects.

Terms: The following terms are used in relation to this example.

Effective dose (ED): It was developed by the ICRP for occupational protection against radiation. ED allows comparing the radiation detriment between a uniform external dose and a non-uniform internal dose. The risk for a DE of 1 rem determined for a non-uniform internal dose is equal to the risk of 1 rem for a uniform external exposure (total body dose), as defined in ICRP publication 60 [ICRP-60 1991].

Effective dose equivalent (EDE acronym): It was developed by the International Commission for Radiological Protection (ICRP) for occupational protection against radiation. The EDE allows to compare the radiation detriment between a dose

247 uniform external and a non-uniform internal dose. The risk for an ED of 1 rem determined for a non-uniform internal dose is equal to the risk of 1 rem for a uniform external exposure (total body dose), as defined in ICRP publication 30 [ICRP-30 1981].

Table 9. All subjects - Estimates of absorbed doses (rem / mCi) n = 12

<td></td><td>Half</td><td>Standard deviation</td><td>Min</td><td>Max</td>

<td>Glands adrenal</td><td> 0.051</td><td> 0.003</td><td> 0.045</td><td> 0.056</td>

<td>Brain</td><td> 0.019</td><td> 0.003</td><td> 0.017</td><td> 0.026</td>

<td>Breasts</td><td> 0.024</td><td> 0.002</td><td> 0.022</td><td> 0.029</td>

<td>Wall of the gallbladder</td><td> 0.059</td><td> 0.005</td><td> 0.050</td><td> 0.069</td>

<td>Wall of lower large intestine</td><td> 0.047</td><td> 0.003</td><td> 0.041</td><td> 0.051</td>

<td>Intestine thin</td><td> 0.170</td><td> 0.029</td><td> 0.121</td><td> 0.215</td>

<td>Stomach wall</td><td> 0.114</td><td> 0.028</td><td> 0.088</td><td> 0.193</td>

<td>Wall of upper large intestine</td><td> 0.059</td><td> 0.005</td><td> 0.051</td><td> 0.066</td>

<td>Wall of heart</td><td> 0.105</td><td> 0.016</td><td> 0.083</td><td> 0.146</td>

<td>Kidneys</td><td> 0.309</td><td> 0.052</td><td> 0.225</td><td> 0.387</td>

<td>Liver</td><td> 0.141</td><td> 0.039</td><td> 0.092</td><td> 0.229</td>

<td>Lungs</td><td> 0.108</td><td> 0.019</td><td> 0.075</td><td> 0.146</td>

<td>Muscle</td><td> 0.030</td><td> 0.002</td><td> 0.028</td><td> 0.035</td>

248

<td>Ovaries</td><td> 0.053</td><td> 0.003</td><td> 0.046</td><td> 0.057</td>

<td>Pancreas</td><td> 0.050</td><td> 0.003</td><td> 0.044</td><td> 0.057</td>

<td>Bone marrow red</td><td> 0.072</td><td> 0.009</td><td> 0.056</td><td> 0.090</td>

<td>Osteogenic cells</td><td> 0.060</td><td> 0.005</td><td> 0.049</td><td> 0.069</td>

<td>Glands salivars</td><td> 0.127</td><td> 0.053</td><td> 0.075</td><td> 0.280</td>

<td>Skin</td><td> 0.020</td><td> 0.002</td><td> 0.019</td><td> 0.025</td>

<td>Spleen</td><td> 0.111</td><td> 0.029</td><td> 0.072</td><td> 0.165</td>

<td>Testicles</td><td> 0.027</td><td> 0.002</td><td> 0.025</td><td> 0.031</td>

<td>Scam</td><td> 0.029</td><td> 0.003</td><td> 0.027</td><td> 0.036</td>

<td>Thyroid</td><td> 0.243</td><td> 0.039</td><td> 0.172</td><td> 0.294</td>

<td>Urinary bladder wall</td><td> 0.376</td><td> 0.073</td><td> 0.179</td><td> 0.463</td>

<td>Uterus</td><td> 0.062</td><td> 0.003</td><td> 0.057</td><td> 0.068</td>

<td>Total body</td><td> 0.038</td><td> 0.002</td><td> 0.036</td><td> 0.043</td>

<td>EDE</td><td> 0.115</td><td> 0.006</td><td> 0.103</td><td> 0.121</td>

<td>FROM</td><td> 0.096</td><td> 0.005</td><td> 0.090</td><td> 0.107</td>

Table 10. All subjects - Estimates of absorbed doses (mSv / MBq) n = 12

<td></td><td>Half</td><td>Standard deviation</td><td>Min</td><td>Max</td>

<td>Kidney glands</td><td> 0.0138</td><td> 0.0009</td><td> 0.0121</td><td> 0.0152</td>

<td>Brain</td><td> 0.0052</td><td> 0.0007</td><td> 0.0046</td><td> 0.0069</td>

<td>Breasts</td><td> 0.0065</td><td> 0.0006</td><td> 0.0060</td><td> 0.0079</td>

<td>Wall of the</td><td> 0.0159</td><td> 0.0014</td><td> 0.0136</td><td> 0.0187</td>

249

<td>gallbladder</td><td></td><td></td><td></td><td></td>

<td>Wall of lower large intestine</td><td> 0.0128</td><td> 0.0007</td><td> 0.0112</td><td> 0.0137</td>

<td>Intestine thin</td><td> 0.0460</td><td> 0.0079</td><td> 0.0327</td><td> 0.0581</td>

<td>Stomach wall</td><td> 0.0308</td><td> 0.0077</td><td> 0.0238</td><td> 0.0520</td>

<td>Wall of upper large intestine</td><td> 0.0159</td><td> 0.0013</td><td> 0.0136</td><td> 0.0178</td>

<td>Wall of heart</td><td> 0.0285</td><td> 0.0043</td><td> 0.0223</td><td> 0.0395</td>

<td>Kidneys</td><td> 0.0834</td><td> 0.0141</td><td> 0.0608</td><td> 0.1046</td>

<td>Liver</td><td> 0.0382</td><td> 0.0104</td><td> 0.0249</td><td> 0.0619</td>

<td>Lungs</td><td> 0.0291</td><td> 0.0053</td><td> 0.0201</td><td> 0.0395</td>

<td>Muscle</td><td> 0.0081</td><td> 0.0005</td><td> 0.0077</td><td> 0.0095</td>

<td>Ovaries</td><td> 0.0143</td><td> 0.0009</td><td> 0.0123</td><td> 0.0155</td>

<td>Pancreas</td><td> 0.0136</td><td> 0.0009</td><td> 0.0120</td><td> 0.0155</td>

<td>Bone marrow red</td><td> 0.0196</td><td> 0.0023</td><td> 0.0150</td><td> 0.0242</td>

<td>Osteogenic cells</td><td> 0.0163</td><td> 0.0015</td><td> 0.0133</td><td> 0.0187</td>

<td>Glands salivars</td><td> 0.0343</td><td> 0.0144</td><td> 0.0204</td><td> 0.0758</td>

<td>Skin</td><td> 0.0055</td><td> 0.0005</td><td> 0.0051</td><td> 0.0068</td>

<td>Spleen</td><td> 0.0300</td><td> 0.0080</td><td> 0.0195</td><td> 0.0446</td>

250

<td>Testicles</td><td> 0.0074</td><td> 0.0005</td><td> 0.0067</td><td> 0.0085</td>

<td>Scam</td><td> 0.0080</td><td> 0.0007</td><td> 0.0074</td><td> 0.0098</td>

<td>Thyroid</td><td> 0.0657</td><td> 0.0106</td><td> 0.0465</td><td> 0.0795</td>

<td>Wall of the urinary bladder</td><td> 0.1015</td><td> 0.0197</td><td> 0.0484</td><td> 0.1251</td>

<td>Uterus</td><td> 0.0169</td><td> 0.0009</td><td> 0.0155</td><td> 0.0183</td>

<td>Total body</td><td> 0.0104</td><td> 0.0004</td><td> 0.0098</td><td> 0.0115</td>

<td>EDE</td><td> 0.0309</td><td> 0.0015</td><td> 0.0278</td><td> 0.0327</td>

<td>FROM</td><td> 0.0260</td><td> 0.0012</td><td> 0.0244</td><td> 0.0288</td>

These data showed that the tracer-1 agent was well tolerated and provided a radiation dose comparable to that of other radiopharmaceuticals commonly used in PET. Myocardial absorption and activity of adjacent organs showed that it was possible to acquire good images with a radiation dose acceptable to the patient.

Example 25

Tracer-1 was designed as a substrate for the norepinephrine transporter (NET) in order to obtain images of the cardiac sympathetic nervous system. Competitive experiments using cell membranes that overexpressed human NET indicated a Ki value of 5.16 ± 0.93 μΜ. In a human neuroblastoma cell line (SH-SY5Y), the absorption of tracer-1 agent was inhibited by desipramine, a selective NET inhibitor, and

251 Absorption kinetics was determined with values ​​of Km and V_max 6.78 + 1.94 pM and 5.18 ± 1.23 pmol / min / million cells, respectively. These values ​​were similar to those of MIBG (2.12 + 0.26 pM and 4.76 ± 0.78 pmol / min / million cells). In animals, tissue biodistribution of tracer-1 agent was evaluated by tissue sampling 15 and 60 minutes after administration. Absorption in the heart was 2.36 ± 0.16 and 2.17 + 0.12% of the dose injected per g of tissue (% of Dl / g) in rats and 0.25 + 0.03 and 0.28 + 0.03% of Dl / g in rabbits. In rabbits, desipramine (1 mg / kg) inhibited the absorption in the heart of tracer-1 by 68% and the absorption of ¹²³I-MIBG 55% 1 hour after the dose. In addition, sympathetic denervation with 6-hydroxypamine (6OHDA, iv) also produced a marked reduction in the absorption of tracer-1 in the heart of 79%. Cardiac tomography with the tracer-1 agent consistently showed a clear picture of the myocardium with minimal background interferences due to blood, lungs or liver in rats, rabbits and nonhuman primates (PNH). In line with biodistribution studies, tomographic studies in rabbits pretreated with desipramine demonstrated reduced levels of radioactivity in the heart in a dose-dependent manner. Similarly, sympathetic denervation induced by 6-OHDA produced a low cardiac image intensity with the tracer-1 agent

252 but normal perfusion images with the PET perfusion agent, (2-tert-butyl-4-chloro-5- [4 - (2 [^ieF] fluoroethoxymethyl) benzyloxy] -2H-pyridazin-3-one (refer to PCT International Publication W02005 / 079391, published September 1, 2005, incorporated herein by reference). Cardiac tomography with tracer-1 agent in PNH pretreated with desipramine (0.5 mg / kg) showed a reduction in radioactivity in the heart of 68%. In vitro and in vivo findings together indicate that tracer-1 agent can be used as a cardiac PET tracer agent transported to the heart through NET and can be used to assess cardiac neuronal function.

Example 26

The prognostic value of the tracer-1 agent was evaluated in salt-sensitive Dahl rats (DSS), a model of heart failure (HF) in rats, and compared with ¹²³Imeta-iodobenzylguanidine (¹²³I-MIBG). DSS rats were fed a low salt diet (0.1% as a control) or a high salt diet (8%). for 5 or 9 weeks. To determine the evolution of HF in these rats, plasma norepinephrine levels, and heart and lung weight were measured. Compared to the groups fed a low salt diet, DSS rats fed a salt diet for 5 weeks had

253 marked increases in norepinephrine levels (258 ± 28 versus 1242 ± 184 pg / mL) and the weight ratio between the heart and body (3.3 ± 0.1 versus 4.5 + 0.3 mg / g). At 9 weeks, the levels of norepinephrine (656 + 219 versus 1508 + 165 pg / mL) and the weight ratio between the heart and body (3.2 + 0.1 versus 6.1 + 0.3 mg / g) had increased further, and the weight ratio between the lungs and the body had increased (3.9 ± 0.1 versus 14.0 + 1.4 mg / g). It was found that these rats fed a salt-rich diet developed HF, from early stage HF with myocardial hypertrophy (5 weeks) to terminal HF with severe pulmonary congestion (9 weeks). Absorption of tracer-1 agent and MIBG in the heart was examined in rats with initial and terminal phase IC taking tissue samples after intravenous administration. Absorption was measured using a gamma counter and expressed as a differential absorption ratio (RAD). The absorption of tracer-1 agent in the heart decreased after the evolution of HF from initial to terminal phase (the group with a low-salt diet versus the group with a high-salt diet: 6.9 ± 0.6 versus 5.1 + 0.6 and 8.1 + 0.2 versus 3.1 + 0.2 RAD after 5 and 9 weeks, respectively). These discoveries are comparable with the absorption of¹²³I-MIBG in the heart of these rats (7.3 + 0.1 vs. 3.8 ± 0.5 and 7.9 + 0.5 vs. 2.3 ± 0.3 RAD, respectively). PET cardiac tomography with the agent

254 Tracer-1 in DSS rats fed a low-salt diet showed a clear picture of the myocardium with minimal background interferences due to blood, lungs or liver. In accordance with the findings of tissue sampling, tomography in DSS rats fed a salt-rich diet showed a progressive reduction in the radioactivity in the heart of these rats between week 5 and week 9. These results suggest that the profile of the tracer-1 agent is similar to that of ¹²³I-MIBG, and that cardiac tomography with the tracer-1 agent can be used to detect the evolution of IC in DSS rats. Example 27

It has been shown that tomography with ¹²³I-metaiodobenzylguanidine (MIBG) predicts the evolution of heart failure, but the image quality is low. Like MIBG, the tracer-1 agent was designed as a substrate for the norepinephrine transporter (NET), but was marked with¹⁸F to take advantage of PET technology. This study evaluated the cardiac image quality of the tracer-1 agent, and its affinity and selectivity for NET and absorption kinetics, compared to norepinephrine (NE).

Methods: Affinity (K¡.) Was determined in a competitive binding assay by incubating ¹⁹F-tracer agent-1, a cold analogue of tracer agent-1, or NE with ³H-desmethylimipramine in

255 a cell membrane that overexpressed human NET. The selectivity of the absorption was evaluated by measuring the cellular absorption of the tracer-1 agent or³H-NE with and without pre-treatment of desipramine, a selective NET inhibitor, in SK-N-SH (human neuroblastoma) and PC-12 (rat pheochromocytoma) cells. In SK-NS cells, the kinetic absorption parameters (Km and V_max) were evaluated by measuring the NET-mediated absorption of tracer-1 or NE at various concentrations. The quality of the cardiac image with the tracer-1 agent was evaluated by PET tomography (-1.5 mCi, iv) in rabbits in the presence and absence of desipramine (1 mg / kg).

Results: In the competitive binding assay, Ki values ​​for tracer-1 and NE were similar (5.2 + 1.1 and 3.4 + 1.3 μΜ). In cell studies, NET blockade inhibited the absorption of tracer-1 agent and NE by 66 + 7 and 93 + 1% in PC-12 cells, and 91 + 1 and 97 ± 1% in SK cells -N-SH. In SK-N-SH cells, the Km and tracer-1 values ​​were 1.4 ± 0.3 μΜ and 6.0 ± 1.3 pMol / million cells / min, these values ​​were similar to those of NE (2.0 + 0.4μΜ and 6.2 ± 0.7 pMol / million cells / min). In addition, cellular absorption of tracer-1 was inhibited by tracer-1 or NE in a concentration-dependent manner. Rabbit tomography with the tracer-1 agent showed a clear picture of the absorption in the

256 myocardium with low liver activity. Cardiac absorption could be inhibited by desipramine.

The cellular absorption profile of the tracer-1 agent was similar to that of NE with high selectivity. Cardiac images of the tracer-1 agent were clear, and absorption in the heart was mediated by NET.

Example 28

The use of cardiac sympathetic denervation (DSC) evaluated by tomography with ¹²³I-metaiodobenzylguanidine (mibg) to predict cardiac events including arrhythmia and death in patients with heart failure (ADMIRE-HF trial). This studio

<td>evaluated the</td><td>tomography with</td><td colspan="2">the agent</td><td colspan="2">plotter-1 for</td>

<td>identify</td><td>DSC</td><td></td><td></td><td></td><td></td>

<td>Methods</td><td>: They were used</td><td>Models</td><td>from</td><td>Regional DSC</td><td>Y</td>

<td colspan="2">Systemic in rabbits. For</td><td>to provoke</td><td>the</td><td>Regional DSC,</td><td>I know</td>

<td>made a</td><td>sternotomy I measured</td><td>to and</td><td>api</td><td>ico phenol (89%</td><td>in</td>

fluid) on the anterior and posterior wall of the left ventricle. To cause systemic denervation, the neurotoxin 6-hydroxypamine (25 tons / kg on days 1, 2, 7 and 8) was administered intravenously. Two weeks after these procedures, tomography of these rabbits was performed with the tracer-1 agent (-1.5 mCi, iv) using a microPET camera for 30 minutes. To ensure that the denervation procedures did not produce changes

257 in the infusion, a tomography of the rabbits was also performed with the perfusion tracer ^1SF (2-tert-butyl4-chloro-5- [4- (2- [^{1 B}F] fluoroethoxymethyl) benzyloxy] -2H-pyridazin3-one).

Results: In rabbits with simulated denervation, the cardiac tracer-1 cardiac images showed a clear image of the myocardium with a uniform radioactivity distribution. Radioactivity was low in the lungs and liver, and was rapidly eliminated in the blood. In rabbits with systemic denervation, image-based quantification indicated ~ 80% overall reduction in cardiac absorption of tracer-1 agent compared to control animals. Similarly, regional denervation resulted in a marked reduction of tracer-1 agent in the treated regions. In contrast, cardiac tomography with (2-tert-butyl-4-chloro-5- [4- (2 [¹⁸F] fluoroethoxymethyl) benzyloxy] -2J / -pyridazin-3-one showed good myocardial perfusion, and no differences were observed between control rabbits and denervated rabbits.

It was determined that the reduction in the absorption of tracer-1 agent in the heart of rabbits with DSC was due to impaired innervation and not perfusion alterations. PET cardiac tomography with tracer-1 agent was used to detect DSC, as well as¹²³I25

MIBG, but with image quality and quantification

258 top .

Example 29

The following example describes the functions of cardiac absorption of norepinephrine 1 and 2 in the evaluation of the tracer-1 agent in non-human rats, rabbits and primates.

Objectives: Norepinephrine (NE) released from cardiac sympathetic nerves is substantially eliminated by neuronal absorption 1 (transporter of NE) in rabbits, nonhuman primates and humans, and by absorption 1 and 2 in rats. Tracer-1 agent is designed, in part, as a substrate for absorption 1 as well as NE and¹²³I-metaiodobenzylguanidine (MIBG). This study examines the differences between species associated with cardiac absorption '1 and 2 for cardiac absorption of tracer-1 agent.

Methods: Desipramine, a selective absorption 1 inhibitor, was used to block cardiac absorption 1 in rats (10 mg / kg, ip), rabbits (1 mg / kg iv) and PNH (0.5 mg / kg, iv). 6-Hydroxypamine, a neurotoxin, was injected to induce sympathetic denervation in rats (100 mg / kg ip for 7 days) and rabbits (25 mg / kg iv on days 1, 2, 7 and 8). The absorption of tracer agent-1 in the heart was evaluated compared to MIBG by taking tissue samples 60 minutes after the tracer agent injection. I also know

259 performed a tomography with (2-tert-butyl-4-chloro-5- [4- (2 [¹⁸F] fluoroethoxymethyl) benzyloxy] -2H-pyridazin-3-one.

Results: In rats, blocking of absorption 1 did not alter the absorption of tracer-1 agent in the heart compared to the control (1.41 + 0.07 versus 1.47 ± 0.22% of the dose injected per gram of tissue (% of DI / g)). In contrast, the absorption of tracer-1 agent in the heart was reduced by 68% in rabbits with absorption 1 blocked. In rats with sympathetic denervation, the absorption of tracer-1 agent in the heart was comparable to the control group (2.18 + 0.39 versus 2.58 ± 0.76% ID / g). However, absorption was markedly reduced (79%) in rabbits with sympathetic denervation. Similar results were obtained for cardiac absorption of MIBG in rats and rabbits with absorption block 1 and sympathetic denervation. According to this, cardiac tomography with (2-tert-butyl-4-chloro-5- [4- (2 [¹⁸F] fluoroethoxymethyl) benzyloxy] -2H-pyridazin-3-one showed comparable myocardial activity in rats with sympathetic denervation, but a marked reduction in activity in rabbits with denervation, and in rabbits and PNH with absorption blockage one.

Conclusions: In rabbits and non-human primates with absorption 1 as the main transporter of cardiac NE, as in humans, the tracer-1 agent showed

260 a high selectivity for neuronal absorption 1 and can be used in the evaluation of cardiac sympathetic denervation. Due to the high cardiac expression of absorption 2, in some embodiments, evaluation of the absorption substrate 1 based on neuronal tracer agents in rats should be carried out with caution. Example 30

The following example describes the evaluation of medications for heart failure based on the cardiac absorption of the tracer-1 agent.

Objectives: This study investigated whether the drugs for HF commonly used affect the absorption of the tracer agent-1 mediated by NET.

Methods: NET-mediated absorption of tracer-1 agent was detected in SK-N-SH cells (human neuroblastoma known to express NET) by incubating 1 million cells with the ligand for 60 minutes in the presence or absence of desipramine (1 μΜ), a selective NET inhibitor. To assess the impact of the drug on the absorption of the tracer-1 agent, the cells were pre-incubated (15 minutes) with vehicle or various concentrations (0.001-1000 μΜ) of propranolol (receptor blocker), captopril (ACE inhibitor), losartan (angiotensin II receptor inhibitor) or verapamil (calcium channel blocker) before adding the tracer-1 agent (1 pCi).

261

Results: The cellular absorption of the tracer-1 agent was 26 + 2% in SK-N-SH cells and most (88%) was inhibited by desipramine. Only a substantial reduction in the absorption of tracer-1 agent was observed for preincubation with propranolol in concentrations greater than 1 μΜ and with verapamil in concentrations greater than 10 μΜ. Losartan and captopril had no effect on the absorption of tracer-1 agent even at the highest concentrations evaluated (1000 μ 1000). The concentrations of these drugs for HF that produced inhibition of the absorption of tracer-1 were substantially higher than the steady-state levels achieved for these drugs in clinical uses.

Conclusions: Based on these in vitro studies, several medications for common-use HF do not inhibit the absorption of the NET-mediated tracer-1 agent at clinically relevant concentrations. Example 31

The following example describes the evaluation of cardiac denervation, reinnervation and propensity for associated arrhythmia using the tracer-1 agent.

Objectives: Regional cardiac sympathetic denervation (DSCR) may be associated with cardiac arrhythmia in patients with heart failure. This study evaluates whether tomography with the tracer-1 agent could be used.

262 to measure the DSCR, the subsequent reinervation and its potential association with the propensity for arrhythmia.

Methods: DSCR models were developed in rabbits by applying phenol directly on the surface of the left ventricular wall during a sternotomy. Two-and twelve weeks after this procedure, PET cardiac tomography was performed with tracer-1 (-1.5 mCi, iv) in these rabbits. The myocardial area with a maximum radioactivity 50% was quantified as an innervated region for comparison. To assess the propensity for arrhythmia in rabbits, changes induced by dofetilide (10 and 40 pg / kg iv, an I inhibitor) were evaluated_Krdelayed) by performing an electrocardiogram (ECG) that included the heart rate (HR), the QTc interval (corrected by the Fridericia method) and the arrhythmia frequency.

Results · The cardiac images showed a clear image of the homogeneous myocardial absorption of tracer-1 agent in rabbits with simulated denervation and reduced levels in rabbits with phenol-induced DSCR 2 weeks after surgery (20702 ± 2190 versus 12245 ± 905 Vóxels, respectively). The denervated region was reduced at 12 weeks (16812 ± 503 voxels) which indicated re-preservation. Tomography with (2-tert-butyl-4-chloro-5- [4 (2- [¹⁸f] fluoroethoxymethyl) benzyloxy] -2H-pyridazin-3-one, a

263 PET perfusion tracing agent showed a homogenous myocardial distribution in all rabbits including those with DSCR regions which indicated that denervation did not alter blood flow. Qtc prolongation, frequency of premature ventricular contraction and polymorphic ventricular tachycardia induced by dofetilide were more prominent in the DSCR group than in the control group. However, the changes in HR were comparable in the two groups.

Conclusions: Cardiac tomography with tracer-1 agent detected DSCR and reinervation. DSCR increased the propensity to prolong QTc and drug-induced arrhythmia.

Example 32

The following example describes the evaluation of the tracer-1 agent in mice suffering from a tumor.

Example 32A

Preparation of models of mice suffering from a tumor

Xenograft model: Female athymic mice aged between 4 and 6 weeks were anesthetized to immobilize them in order to inoculate them subcutaneously with a range between 1.0 x 10⁶/0.1 cells / mL · and 1.0 X 10⁸/0.1 cells / mL in sterile cell culture media, and then reintroduced into their cages to recover. Cell lines were co-injected with a

264 culture matrix that can be purchased from commercial suppliers (50/50 v / v) to facilitate tumor development (Matrigel®-BD Bioscience). Human cell lines included PC12 (pheochromocytoma), SH-SY-5Y and SK-N-SH (neuroblastoma).

Oncomouse model: Obtained through an internal selective breeding program.

Example 32B

Tissue biodistribution

Mice suffering from tumors were anesthetized (100-1500 mm³ tumor size) intramuscularly with 0.1 mL of ketamine / acepromazine (1.8 mL of saline solution, 1.0 mL of ketamine and 0.2 mL of acepromazine) before administering doses and taking tissue samples. Next, the tracer-1 agent (0.5-2.0 mCi / kg in 0.1 mL) was injected into individual mice through the tail vein. Mice were sacrificed and biodistribution was performed 1 h after injection. The selected tissues were excised, weighed and analyzed in a gamma counter. The results were expressed as the percentage of dose injected per gram of tissue (% ID / g; Figure 10). Because c-neu Oncomouse® mice develop tumors spontaneously in the mammary glands, most mice developed more than one tumor. Samples of each tumor were taken, analyzed separately and the

265 average radioactivity absorption for tumors in order to obtain a global representation of tumor absorption. Mice with xenografts had only one tumor implanted and removed at the time of analysis of tissue distribution.

Terms and equivalents

Although various embodiments of the present invention have been described and illustrated herein, those skilled in the art will be able to easily determine several different methods and / or structures for carrying out the functions and / or obtaining the results and / or one or more of the advantages described herein; It will be considered that each of these variations and / or modifications is encompassed within the scope of the present invention. In more general terms, those skilled in the art will readily realize that all parameters, dimensions, materials and configurations described herein are intended to be examples and that the actual parameters, dimensions, materials and / or configurations will depend of the application or specific applications for for which the knowledge of the present invention will be used. Those skilled in the art will recognize or be able to determine, using only routine experiments, many equivalents of the specific embodiments of the invention described herein. Therefore, it

266 It will be understood that the preceding embodiments are presented by way of example only and that, within the scope of the appended claims and their equivalents, the invention can be practiced in a manner other than that claimed and specifically described. The present invention relates to each feature, system, article, material, kit and / or individual method described herein. In addition, any combination of two or more of these characteristics, systems, articles, materials, kits and / or methods is included in the scope of the present invention, provided that these characteristics, systems, articles, materials, kits and / or methods are not Be mutually inconsistent.

It should be understood that the indefinite articles one and one, as used herein in the description and in the claims, mean at least one, unless clearly stated otherwise.

It should be understood that the expression and / or, as used herein in the description and in the claims, means all or one of the elements joined in this way, that is, the elements that are present conjunctively in some cases and disjunctively in other cases. Optionally there may be other elements present other than the elements specifically identified by the clause and / or, both related

267 as not related to the specifically identified elements, unless clearly stated otherwise. Thus, as a non-limiting example, a reference to A and / or B, when used in conjunction with an open expression such as comprising, may refer to, in one embodiment, A without B (which optionally includes elements other than B) ; in another embodiment, to B without A (which optionally includes elements other than A); in another embodiment, both A and B (which optionally includes other elements); etc.

It should be understood that or, as used herein in the description and in the claims, has the same meaning as and / or defined above. For example, when separating elements in a list, o and / or should be interpreted as inclusive, that is, they include at least one, but also include more than one, from a number or list of elements and, optionally, additional elements not listed Only terms that clearly indicate otherwise, such as only one of or exactly one of or, when used in the claims, consisting of, will refer to the inclusion of exactly one element of a number or a list of elements. In general, it should be interpreted that the term or, as used herein, indicates only exclusive alternatives (i.e. one or the other

268 but not both) when preceded by terms of exclusivity such as one of the, one of, only one of or exactly one of. The expression consisting essentially of, when used in the claims, should be given its usual meaning used in the field of patent law.

It should be understood that the expression at least one, as used herein in the description and in the claims, when referring to a list of one or more elements means at least one element selected from any one or more elements of the item list, but does not necessarily include at least one of each and every item listed specifically in the item list, and does not exclude any combinations of elements in the list of elements. This definition also allows that there may optionally be different elements present from the elements specifically identified in the list of elements to which the expression refers to at least one, both related and unrelated to the specifically identified elements. Thus, as a non-limiting example, at least one of the elements A and B (or, equivalently, at least one of the elements A or B or, equivalently, at least one of the elements A and / or B ) may refer, in one modality, to at least one, which optionally includes more than

269 a, element A, without B present (and optionally including elements other than B); in another embodiment, at least one, which optionally includes more than one, element B, without A present (and optionally including elements other than A); in another embodiment, at least one, which optionally includes more than one, element A and at least one, which optionally includes more than one, element B (and optionally includes other elements); etc.

In the claims, as well as in the previous description, it should be understood that all connective expressions such as comprising, holding, holding, containing, implying, maintaining and similar are open expressions, that is to say , mean that includes but not limited to. Only the connective expressions composed of and composed essentially of shall be closed or semi-closed connective expressions, respectively, as set forth in Section 2111.03 of the Manual of Procedures for the Patent Examination of the United States Patent Office.

It is noted that in relation to this date, the best method known by the applicant to implement said invention is that which is clear from the present description of the invention.