Pyrazine derivatives and uses thereof in renal monitoring

Abstract

The present invention relates to pyrazine derivatives such as those represented by Formulas I and II below.

##STR00001##
X¹ to X⁴ of the compounds of Formulas I and II may be characterized as electron withdrawing groups. In contrast, Y¹ to Y⁴ of the compounds of Formulas I and II may be characterized as electron donating groups. Pyrazine derivatives of the present invention may be utilized in assessing renal function. In particular, an effective amount of a pyrazine derivative of the invention may be administered into a body of a patient. The pyrazine derivative that is in the body may be exposed to visible and/or infrared light to cause spectral energy to emanate from the pyrazine derivative. This emanating spectral energy may be detected and utilized to determine renal function of the patient.

Description

REFERENCE TO RELATED APPLICATIONS

This application is a —CONH(AA)—CONH(AA) —CO(AA), and —CONH(PS), wherein at least one of (e.g., one of or both of) X¹ and X² is independently either —CONH(AA) —CO(AA) or —CONH(PS). For instance, in one groups of embodiments, at least one of X¹ and X² is —CONH(AA) —CO(AA). In another group of embodiments, at least one of X¹ and X² is —CONH(PS). With further regard to this first family of embodiments, Y¹ and Y² are independently selected from the group consisting of —OR¹⁰, —SR¹¹, —NR¹²R¹³, —N(R¹⁴)COR¹⁵, —P(R¹⁶)₃, —P(OR¹⁷)₃, and substituents corresponding to Formula A above.

In some embodiments of this first family, X¹ and X² are independently selected from the group consisting of —CN, —CO₂R¹, —CONR²R³, —CONH(AA) —CO(AA), and —CONH(PS), wherein at least one of X¹ and X² is independently either —CONH(AA) —CO(AA) or —CONH(PS). Y¹ and Y² of some embodiments of the first family are independently selected from the group consisting of —NR¹²R¹³ and substituents corresponding to Formula A above.

In a second family of embodiments, X¹ and X² are independently selected from the group consisting of —CN, —CO₂R¹, —CONR²R³, —COR⁴, —NO₂, —SOR⁵, —SO₂R⁶, —SO₂OR⁷, —PO₃R⁸R⁹, —CONH(AA) —CO(AA), and —CONH(PS). Further, Y¹ and Y² are independently selected from the group consisting of —OR¹⁰, —SR¹¹, —NR¹²R¹³, —N(R¹⁴)COR¹⁵, —P(R¹⁶)₃, —P(OR¹⁷)₃, and substituents corresponding to Formula A above, wherein at least one of (e.g., one of or both of) Y¹ and Y² is independently either —P(R¹⁶)₃ or —P(OR¹⁷)₃. For instance, in one group of embodiments, at least one of Y¹ and Y² is —P(R¹⁶)₃. In another group of embodiments, at least one of Y¹ and Y² is —P(OR¹⁷)₃.

In some embodiments of this second family, X¹ and X² are independently selected from the group consisting of —CN, —CO₂R¹, —CONR²R³; —CONH(AA) —CO(AA), and —CONH(PS). In some embodiments of the second family, one of Y¹ and Y² is —P(R¹⁶)₃ or —P(OR¹⁷)₃, and the other of Y¹ and Y² is —NR¹²R¹³ or a substituent corresponding to Formula A above.

With regard to the above-described first and second families, Z¹ is selected from the group consisting of a single bond, —CR¹⁸R¹⁹, —O, —NR²⁰, —NCOR²¹, —S, —SO and —SO₂. In some embodiments, Z¹ is selected from the group consisting of —O, —NR²⁰, —S, —SO, and —SO₂. In other embodiments, Z¹ is selected from the group consisting of —O and —NR²⁰.

R¹ to R²¹ of the first and second families are independently selected from the group consisting of —H, —(CH₂)_aOR⁴³, —CH₂(CHOH)_aR⁴⁴, —CH₂(CHOH)_aCO₂H, —(CHCO₂H)_aCO₂H, —(CH₂)_aNR⁴⁵R⁴⁶, —CH[(CH₂)_bNH₂]_aCO₂H, —CH[(CH₂)_bNH₂]_aCH₂OH, —CH₂(CHNH₂)_aCH₂NR⁴⁷R⁴⁸, —(CH₂CH₂O)_cR⁴⁹, —(CH₂)_dCO(CH₂CH₂O)_cR⁵⁰, —(CH₂)_aSO₃H, —(CH₂)_aSO₃⁻, —(CH₂)_aOSO₃H, —(CH₂)_aOSO₃⁻, —(CH₂)_aNHSO₃H, —(CH₂)_aNHSO₃⁻, —(CH₂)_aPO₃H₂, —(CH₂)_aPO₃H⁻, —(CH₂)_aPO₃⁼, —(CH₂)_aOPO₃H₂, —(CH₂)_aOPO₃H⁻ and —(CH₂)_aOPO₃. In such embodiments, R⁴³ to R⁵⁰ are independently —H or —CH₃. In one group of embodiments, R¹ to R²¹ of the first and second families are independently selected from the group consisting of —H, —(CH₂)_aOR⁴³, —CH₂(CHOH)_aR⁴⁴, —CH₂(CHOH)_aCO₂H, —(CHCO₂H)_aCO₂H, —(CH₂)_aNR⁴⁵R⁴⁶, —CH[(CH₂)_bNH₂]_aCO₂H, —CH[(CH₂)_bNH₂]_aCH₂OH, —CH₂(CHNH₂)_aCH₂NR⁴⁷R⁴⁸, —(CH₂CH₂O)_cR⁴⁹, —(CH₂)_dCO(CH₂CH₂O)_cR⁵⁰. In another group of embodiments, R¹ to R²¹ are independently selected from the group consisting of —H, —(CH₂)_aOR⁴³, —CH₂(CHOH)_aR⁴⁴, —(CH₂)_aNR⁴⁵R⁴⁶, —(CH₂CH₂O)_cR⁴⁹, and —(CH₂)_dCO(CH₂CH₂O)_dR⁵⁰. In still another group of embodiments, R¹ to R²¹ are independently selected from the group consisting of —H, —(CH₂)_aOR⁴³, —CH₂(CHOH)_aR⁴⁴, —(CH₂)_aNR⁴⁵R⁴⁶, and —(CH₂)_dCO(CH₂CH₂O)_dR⁵⁰.

Still with regard to these first and second families, ‘a’, ‘b’, and ‘d’ independently vary from 1 to 10, ‘c’ varies from 1 to 100, and ‘m’ and ‘n’ independently varies from 1 to 3. In some embodiments, each of ‘a’, ‘b’, and ‘d’ independently varies from 1 to 6. In some embodiments, ‘c’ varies from 1 to 20. In some embodiments, ‘m’ and ‘n’ are independently 0 or 1.

(AA) is a single natural or unnatural amino acid of a polypeptide chain including one two or more natural or unnatural α-amino acids linked together by peptide bonds. The polypeptide chain (AA) may be a homopolypeptide chain or a heteropolypeptide chain, and may be any appropriate length. For instance, in some embodiments, the polypeptide chain may include 1 2 to 100 α-amino acid(s), 1 2 to 90 α-amino acid(s), 1 2 to 80 α-amino acid(s), 1 2 to 70 α-amino acid(s), 1 2 to 60 α-amino acid(s), 1 2 to 50 α-amino acid(s), 1 2 to 40 α-amino acid(s), 1 2 to 30 α-amino acid(s), 1 2 to 20 α-amino acid(s), or even 1 2 to 10 α-amino acid(s). In some embodiments, the α-amino acids of the polypeptide chain (AA) are selected from the group consisting of aspartic acid, asparigine, arginine, histidine, lysine, glutamic acid, glutamine, serine, and homoserine. In some embodiments, the α-amino acids of the polypeptide chain (AA) are selected from the group consisting of aspartic acid, glutamic acid, serine, and homoserine. In some embodiments, the polypeptide chain (AA) refers to a single amino acid (e.g., either aspartic acid or serine).

(PS) is a sulfated or non-sulfated polysaccharide chain including one or more monosaccharide units connected by glycosidic linkages. The polysaccharide chain (PS) may be any appropriate length. For instance, in some embodiments, the polysaccharide chain may include 1 to 100 monosaccharide unit(s), 1 to 90 monosaccharide unit(s), 1 to 80 monosaccharide unit(s), 1 to 70 monosaccharide unit(s), 1 to 60 monosaccharide unit(s), 1 to 50 monosaccharide unit(s), 1 to 40 monosaccharide unit(s), 1 to 30 monosaccharide unit(s), 1 to 20 monosaccharide unit(s), or even 1 to 10 monosaccharide unit(s). In some embodiments, the polysaccharide chain (PS) is a homopolysaccharide chain consisting of either pentose or hexose monosaccharide units. In other embodiments, the polysaccharide chain (PS) is a heteropolysaccharide chain consisting of one or both pentose and hexose monosaccharide units. In some embodiments, the monosaccharide units of the polysaccharide chain (PS) are selected from the group consisting of glucose, fructose, mannose, xylose and ribose. In some embodiments, the polysaccharide chain (PS) refers to a single monosaccharide unit (e.g., either glucose or fructose).

##STR00005##

The present invention is also includes pyrazine derivatives corresponding to Formula II above. In a third family of embodiments of the invention, X³ and X⁴ are independently selected from the group consisting of —CN, —CO₂R²², —CONR²³R²⁴, —COR²⁵, —NO₂, —SOR²⁶, —SO₂R²⁷, —SO₂OR²⁸, —PO₃R²⁹R³⁰, —CONH(AA) —CO(AA), and —CONH(PS), wherein at least one of (e.g., one of or both of) X³ and X⁴ is independently either —CONH(AA) —CO(AA) or —CONH(PS). For instance, in one groups of embodiments, at least one of X³ and X⁴ is —CONH(AA) —CO(AA). In another group of embodiments, at least one of X³ and X⁴ is —CONH(PS). With further regard to this first family of embodiments, Y³ and Y⁴ are independently selected from the group consisting of —OR³¹, —SR³², —NR³³R³⁴, —N(R³⁵)COR³⁶, —P(R³⁷)₃, —P(OR³⁸)₃, and substituents corresponding to Formula B above.

In some embodiments of this third family, X³ and X⁴ are independently selected from the group consisting of —CN, —CO₂R²², —CONR²³R²⁴, —CONH(AA)_r —CO(AA)_r, and —CONH(PS)_s, wherein at least one of X³ and X⁴ is independently either —CONH(AA) —CO(AA) or —CONH(PS). Y³ and Y⁴ of some embodiments of the third family are independently selected from the group consisting of —NR³¹R³² and substituents corresponding to Formula B above.

In still a fourth family of embodiments, X³ and X⁴ are independently selected from the group consisting of —CN, —CO₂R²², —CONR²³R²⁴, —COR²⁵, —NO₂, —SOR²⁶, —SO₂R²⁷, —SO₂OR²⁸, —PO₃R²⁹R³⁰, —CONH(AA) —CO(AA), and —CONH(PS). Further, Y³ and Y⁴ are independently selected from the group consisting of —OR³¹, —SR³², —NR³³R³⁴, —N(R³⁵)COR³⁶, —P(R³⁷)₃, —P(OR³⁸)₃, and substituents corresponding to Formula B above, wherein at least one of Y³ and Y⁴ is independently either —P(R³⁷)₃ or —P(OR³⁸)₃. For instance, in one group of embodiments, at least one of Y³ and Y⁴ is —P(R³⁷)₃. In another group of embodiments, at least one of Y³ and Y⁴ is —P(OR³⁸)₃.

In some embodiments of this fourth family, X³ and X⁴ are independently selected from the group consisting of —CN, —CO₂R²², —CONR²³R²⁴, —CONH(AA) —CO(AA), and —CONH(PS). In some embodiments of the fourth family, one of Y³ and Y⁴ is —P(R³⁷)₃ or —P(OR³⁸)₃, and the other of Y³ and Y⁴ is —NR³¹R³² or a substituent corresponding to Formula B above.

With regard to the above-described third and fourth families, Z² is selected from the group consisting of a single bond, —CR³⁹R⁴⁰, —O, —NR⁴¹, —NCOR⁴², —S, —SO, and —SO₂. In some embodiments, Z² is selected from the group consisting of —O, —NR⁴¹, —S, —SO and —SO₂. In some embodiments, Z² is selected from the group consisting of —O and —NR⁴¹.

R²² to R⁴² are independently selected from the group consisting of —H, —(CH₂)_eOR⁵¹, —CH₂(CHOH)_eR⁵², —CH₂(CHOH)_eCO₂H, —(CHCO₂H)_eCO₂H, —(CH₂)_eNR⁵³R⁵⁴, —CH[(CH₂)_fNH₂]_eCO₂H, —CH[(CH₂)_fNH₂]_eCH₂OH, —CH₂(CHNH₂)_eCH₂NR⁵⁵R⁵⁶, —(CH₂CH₂O)_gR⁵⁷, —(CH₂)_hCO(CH₂CH₂O)_gR⁵⁸, —(CH₂)_eSO₃H, —(CH₂)_eSO₃⁻, —(CH₂)_eOSO₃H, —(CH₂)_eOSO₃⁻, —(CH₂)_eNHSO₃H, —(CH₂)_eNHSO₃⁻, —(CH₂)_ePO₃H₂, —(CH₂)_ePO₃H⁻, —(CH₂)_ePO₃⁼, —(CH₂)_eOPO₃H₂, —(CH₂)_eOPO₃H⁻ end —(CH₂)_eOPO₃. In such embodiments, R⁵¹ to R⁵⁸ are independently —H or —CH₃. In one group of embodiments, R²² to R⁴² are independently selected from the group consisting of —H, —(CH₂)_eOR⁵¹, —CH₂(CHOH)_eR⁵², —CH₂(CHOH)_eCO₂H, —(CHCO₂H)_eCO₂H, —(CH₂)_eNR⁵³R⁵⁴, —CH[(CH₂)_fNH₂]_eCO₂H, —CH[(CH₂)_fNH₂]_eCH₂OH, —CH₂(CHNH₂)_eCH₂NR⁵⁵R⁵⁶, —(CH₂CH₂O)_gR⁵⁷, and —(CH₂)_hCO(CH₂CH₂O)_gR⁵⁸. In another group of embodiments, R²² to R⁴² are independently selected from the group consisting of —H, —(CH₂)_eOR⁵¹, —CH₂(CHOH)_eR⁵², —(CH₂)_eNR⁵³R⁵⁴, —(CH₂CH₂O)_gR⁵⁷, and —(CH₂)_hCO(CH₂CH₂O)_gR⁵⁸. In still another group of embodiments, R²² to R⁴² are independently selected from the group consisting of —H, —(CH₂)_eOR⁵¹, —CH₂(CHOH)_eR⁵², —(CH₂)_eNR⁵³R⁵⁴, and —(CH₂)_hCO(CH₂CH₂O)_gR⁵⁸.

Still with regard to the third and fourth families, ‘e’, ‘f’, and ‘h’ independently vary from 1 to 10, ‘g’ varies from 1 to 100, and ‘p’ and ‘q’ independently vary from 1 to 3. In some embodiments, ‘e’, ‘f’, and ‘h’ independently vary from 1 to 6. In some embodiments, ‘g’ varies from 1 to 20. In some embodiments, ‘m’ and ‘n’ are independently 0 or 1.

As with the first and second families of embodiments described above, (AA) of the third and fourth families is a single natural or unnatural amino acid or a polypeptide chain including one two or more natural or unnatural α-amino acids linked together by peptide bonds. Accordingly, the description of (AA) with reference to the first and second families of embodiments above applies to (AA) of the third and fourth families of embodiments as well. Likewise, (PS) of the third and fourth families is a sulfated or non-sulfated polysaccharide chain including one or more monosaccharide units connected by glycosidic linkages. As such, the description of (PS) with reference to the first and second families of embodiments above applies to (PS) of the third and fourth families of embodiments as well.

Syntheses of pyrazine derivatives, in general, have been previously studied [27] and described [25, 26, 28, 29]. Preparation procedures for some of the pyrazine derivatives of the present invention, using procedures similar to the above references, are described later in Examples 1 to 11. It is noteworthy that the alkylation of the electron donating amino group in cyano- or carboxypyrazines has a profound effect on electronic transition of the pyrazine chromophore in that the dialkylation of the amino group in 2,5-diamino-3,5-dicyanopyrazine produces large bathochromic shift of about 40-60 nm. It is also noteworthy that the pyrrolidino and piperidio derivatives exhibit substantial difference in their UV spectra in that the former exhibits a bathochromic shift of about 34 nm. These results could be explained on the basis that the highest occupied molecular orbital (HOMO) of the alkylated aminopyrazine is destabilized compared to the parent amino compound. Therefore, based on the above premise, it is predicted that pyrazine derivatives containing highly strained azacycloalkyl substituents, which were not disclosed previously, exhibit larger bathochromic shifts compared to unstrained cyclic analogs.

In accordance with the present invention, one protocol for assessing physiological function of body cells includes administering an effective amount of a pyrazine derivative represented by Formula I or II into a body of a patient. An appropriate dosage of the pyrazine derivate that is administered to the patient is readily determinable by one of ordinary skill in the art and may vary according to the clinical procedure contemplated, generally ranging from about 1 nanomolar to about 100 micromolar. The administration of the pyrazine derivative to the patient may occur in any of a number of appropriate fashions including, but not limited to: (1) intravenous, intraperitoneal, or subcutaneous injection or infusion; (2) oral administration; (3) transdermal absorption through the skin; and (4) inhalation.

Pyrazine derivatives of this invention can be administered as solutions in most pharmaceutically acceptable intravenous vehicles known in the art. Pharmaceutically acceptable vehicles that are well known to those skilled in the art include, but are not limited to, 0.01-0.1 M phosphate buffer or 0.8% saline. Additionally, pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, emulsions, or appropriate combinations thereof. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Examples of aqueous carriers are water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Exemplary parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Exemplary intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, collating agents, inert gases and the like.

Suitable diluents, preservatives, solubilizers, emulsifiers, adjuvant and/or carriers are also suitable excipients. Such compositions are liquids or lyophilized or otherwise dried formulations and include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), solubilizing agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), bulking substances or tonicity modifiers (e.g., lactose, mannitol), complexation with metal ions, or incorporation of the material into or onto particulate preparations of polymeric compounds such as polylactic acid, polglycolic acid, hydrogels, etc, or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Such compositions may likely influence the physical state, solubility, stability, rate of in vivo release, and/or rate of in vivo clearance.

Still referring to the above-mentioned protocol, the pyrazine derivative is exposed to visible and/or near infrared light. This exposure of the pyrazine derivate to light may occur at any appropriate time but preferably occurs while the pyrazine derivative is located in the body. Due to this exposure of the pyrazine derivate to the visible and/or infrared light, the pyrazine derivate emanates spectral energy (e.g., visible and/or near infrared light) that may be detected by appropriate detection equipment. The spectral energy emanated from the pyrazine derivative tends to exhibit a wavelength range greater than a wavelength range absorbed by the pyrazine derivative. For example, if an embodiment of the pyrazine derivative absorbs light of about 700 nm, the pyrazine derivative may emit light of about 745 nm.

Detection of the pyrazine derivate (or more particularly, the light emanating therefrom) may be achieved through optical fluorescence, absorbance or light scattering procedures known in the art. In one embodiment, this detection of the emanated spectral energy may be characterized as a collection of the emanated spectral energy and a generation of electrical signal indicative of the collected spectral energy. The mechanism(s) utilized to detect the spectral energy from the pyrazine derivative that is present in the body may be designed to detect only selected wavelengths (or wavelength ranges) and/or may include one or more appropriate spectral filters. Various catheters, endoscopes, ear clips, hand bands, head bands, surface coils, finger probes and the like may be utilized to expose the pyrazine derivative to light and/or to detect the light emanating therefrom [30]. This detection of spectral energy may be accomplished at one or more times intermittently or may be substantially continuous.

Renal function of the patient can be determined based on the detected spectral energy. This can be achieved by using data indicative of the detected spectral energy and generating an intensity/time profile indicative of a clearance of the pyrazine derivative from the body. This profile may be correlated to a physiological or pathological condition. For example, the patient's clearance profiles and/or clearance rates may be compared to known clearance profiles and/or rates to assess the patient's renal function and to diagnose the patient's physiological condition. In the case of analyzing the presence of the pyrazine derivative in bodily fluids, concentration/time curves may be generated and analyzed (preferably in real time) using an appropriate microprocessor to diagnose renal function.

Physiological function can be assessed by: (1) comparing differences in manners in which normal and impaired cells remove a pyrazine derivative of the invention from the bloodstream; (2) measuring a rate or an accumulation of a pyrazine derivative of the invention in the organs or tissues; and/or (3) obtaining tomographic images of organs or tissues having a pyrazine derivative of the invention associated therewith. For example, blood pool clearance may be measured non-invasively from convenient surface capillaries such as those found in an ear lobe or a finger or can be measured invasively using an appropriate instrument such as an endovascular catheter. Accumulation of a pyrazine derivative of the invention within cells of interest can be assessed in a similar fashion.

A modified pulmonary artery catheter may also be utilized to, inter alia, make the desired measurements [32] of spectral energy emanating from a pyrazine derivative of the invention. The ability for a pulmonary catheter to detect spectral energy emanating from a pyrazine derivative of the invention is a distinct improvement over current pulmonary artery catheters that measure only intravascular pressures, cardiac output and other derived measures of blood flow. Traditionally, critically ill patients have been managed using only the above-listed parameters, and their treatment has tended to be dependent upon intermittent blood sampling and testing for assessment of renal function. These traditional parameters provide for discontinuous data and are frequently misleading in many patient populations.

Modification of a standard pulmonary artery catheter only requires making a fiber optic sensor thereof wavelength-specific. Catheters that incorporate fiber optic technology for measuring mixed venous oxygen saturation exist currently. In one characterization, it may be said that the modified pulmonary artery catheter incorporates a wavelength-specific optical sensor into a tip of a standard pulmonary artery catheter. This wavelength-specific optical sensor can be utilized to monitor renal function-specific elimination of a designed optically detectable chemical entity such as the pyrazine derivatives of the present invention. Thus, by a method analogous to a dye dilution curve, real-time renal function can be monitored by the disappearance/clearance of an optically detected compound.

The following examples illustrate specific embodiments of the invention. As would be apparent to skilled artisans, various changes and modifications are possible and are contemplated within the scope of the invention described.

EXAMPLE 1

Preparation of 3,6-diamino-N²,N²,N⁵,N⁵-tetrakis(2-methoxyethyl)pyrazine-2,5-dicarboxamide

##STR00006##

A mixture of 3,6-diaminopyrazine-2,5-dicarboxylic acid (200 mg, 1.01 mmol), bis-2-(methoxyethyl)amine (372 μL, 335.5 mg, 2.52 mmol), HOBt-H₂O (459 mg, 3.00 mmol), and EDC-HCl (575 mg, 3.00 mmol) were stirred together in DMF (20 mL) for 1 h at room temperature. The mixture was concentrated to dryness and the residue was partitioned with EtOAc and water. The layers were separated and the EtOAc solution was washed with saturated NaHCO₃ and brine. The solution was dried over anhydrous Na₂SO₄, filtered and concentrated. Purification by radial flash chromatography (SiO₂, 10/1 CHCl₃-MeOH) afforded 228.7 mg (53% yield) of Example 1 as an orange foam: ¹H NMR (300 MHz, CDCl₃), δ 4.92 (s, 4H), 3.76 (apparent t, J=5.4 Hz, 4H), 3.70 (apparent t, J=5.6 Hz, 4H), 3.64 (apparent t, J=5.4 Hz, 4H), 3.565 (apparent t, J=5.4 Hz), 3.67 (s, 6H), 3.28 (s, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 167.6 (s), 145.6 (s), 131.0 (s), 72.0 (t), 70.8 (t), 59.2 (q), 49.7 (t), 47.1 (t). LCMS (5-95% gradient acetonitrile in 0.1% TFA over 10 min), single peak retention time=3.14 min on 30 mm column, (M+H)⁺=429. UV/vis (100 μM in PBS) λ_abs=394 nm. Fluorescence (100 nm) λ_ex=394 nm λ_em=550 nm.

EXAMPLE 2

3,6-diamino-N²,N⁵-bis(2,3-dihydroxydrodyl)dyrazine-2,5-dicarboxamide

##STR00007##

Step 1. Synthesis of 3,6-diamino-N²,N⁵-bis((2,2-dimethyl-1,3-dioxolan-4-yl)methyl)pyrazine-2,5-dicarboxamide

##STR00008##

A mixture of 3,6-diaminopyrazine-2,5-dicarboxylic acid (350 mg, 1.77 mmol), racemic (2,2-dimethyl-1,3-dioxolan-4-yl)methanamine (933 μL, 944 mg, 7.20 mmol), HOBt-H₂O (812 mg, 5.3 mmol), and EDC-HCl (1.02 g, 5.32 mmol) were stirred together in DMF (20 mL) for 16 h at room temperature. The mixture was concentrated to dryness and the residue was partitioned with EtOAc and water. The layers were separated and the EtOAc solution was washed with saturated NaHCO₃ and brine. The solution was dried over anhydrous Na₂SO₄, filtered and concentrated to afford 665 mg (88% yield) of the bis-amide diastereomeric pair as a yellow solid: ¹NMR (300 MHz, CDCl₃) δ 8.38 (t, J=5.8 Hz, 2H), 6.55 (s, 4H), 4.21 (quintet, J=5.8 Hz, 2H), 3.98 (dd, J=8.4 Hz, 6.3 Hz, 2H), 3.65 (dd, J=8.4 Hz, J=5.8 Hz, 2H), 3.39 (apparent quartet—diastereotopic mixture, J=5.9 Hz, 4H), 1.35 (s, 6H), 1.26 (s, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 165.7 (s), 146.8 (s), 126.8 (s), 109.2 (s), 74.8 (d), 67.2 (t), 42.2, 41.1 (t—diastereotopic pair), 27.6 (q), 26.1 (q).

Step 2

The product from Step 1 was dissolved in THF (100 mL) and treated with 1.0 N HCl (2 mL). After hydrolysis was complete, the mixture was treated with K₂CO₃ (1 g) and stirred for 1 h and filtered through a plug of C18 with using methanol. The filtrate was concentrated to dryness and the residue was triturated with MeOH (50 mL). The solids were filtered and discarded and the residue was treated with ether (50 mL). The precipitate was collected by filtration and dried at high vacuum. This material was purified by radial flash chromatography to afford 221 mg (36% yield) of Example 2 as a orange solid: ¹NMR (300 MHz, DMSO-d₆) δ 8.00 (bm, 6H), 5.39 (bs, 2H), 4.88 (bs, 2H), 3.63-3.71 (complex m, 2H), 3.40 (dd, J=11.1, 5.10 Hz, 2H), 3.28 (dd, J=11.1, 6.60 Hz, 2H), 2.92 (dd, J=12.6, 3.3 Hz, 2H), 2.65 (dd, J=12.6, 8.4 Hz, 2H). LCMS (5-95% gradient acetonitrile in 0.1% TFA over 10 min), single peak retention time=4.13 min on 30 mm column, (M+H)⁺=345. UV/vis (100 μM in H₂O) λ_abs=432 nm. Fluorescence λ_ex=432 nm, λ_em=558 nm.

EXAMPLE 3

3,6-Diamino-N²,N⁵-bis(serine)-pyrazine-2,5-dicarboxamide

##STR00009##

Step 1. Synthesis of 3,6-Diamino-N²,N⁵-bis(O-benzylserine methyl ester)-pyrazine-2,5-dicarboxamide

##STR00010##

A mixture of sodium 3,6-diaminopyrazine-2,5-dicarboxylate (300 mg, 1.24 mmol), Ser(OBn)-OMe-HCl salt (647 mg, 2.64 mmol), HOBt-H₂O (570 mg, 3.72 mmol) and EDC-HCl (690 mg, 3.60 mmol) in DMF (25 mL) was treated with TEA (2 mL). The resulting mixture was stirred for 16 h and concentrated. Work up as in Example 1 afforded 370 mg (51% yield) of the bisamide as a bright yellow powder: ¹NMR (300 MHz, CDCl₃) δ 8.47 (d, J=8.74 Hz, 2H), 7.25-7.37 (complex m, 10H), 5.98 (bs, 4H), 4.85 (dt, J=8.7, 3.3 Hz, 2H), 4.56 (ABq, J=12.6, Hz, Δν=11.9 Hz, 4H), 3.99 (one half of an ABq of d, J=8.7, 3.3, Δν obscured, 2H), 3.76-3.80 (one half of an ABq obscured, 2H), 3.78 (s, 6H). 13C NMR (75 MHz, CDCl3) 170.5 (s), 165.1 (s), 146.8 (s), 138.7 (s) 128.6 (d), 128.1 (d), 127.8 (d), 126.9 (s), 73.5 (t), 69.8 (t), 53.0 (q), 52.9 (q). LCMS (5-95% gradient acetonitrile in 0.1% TFA over 10 min), single peak retention time=4.93 min on 30 mm column, (M+H)+=581.

Step 2. Synthesis of 3,6-Diamino-N²,N⁵-bis(O-benzylserine)-pyrazine-2,5-dicarboxamide

##STR00011##

The product from step 1 (370 mg, 0.64 mmol) in THF (10 mL) was treated with 1.0 N sodium hydroxide (2.5 mL). After stirring at room temperature for 30 min, the reaction was judged complete by TLC. The pH was adjusted to approximately 2 by the addition of 1.0 N HCl and the resulting solution was extracted (3×) with EtOAc. The layers were combined, dried over sodium sulfate, filtered and concentrated to afford 353 mg (100% yield) of the diacid as an orange foam: LCMS (5-95% gradient acetonitrile in 0.1% TFA over 10 min), retention time=4.41 min on 30 mm column, (M+H)⁺=553.

Step 3

To the product from step 2 (353 mg, 0.64 mmol) in methanol (20 mL) was added 5% Pd/C (300 mg) and ammonium formate (600 mg). The resulting reaction was heated at reflux for 2 h. The reaction was cooled to room temperature, filtered through a plug of celite and concentrated. The residue was recrystallized from methanol-ether to provide 191 mg (80% yield) of Example 3 as a yellow foam: ¹NMR (300 MHz, DMSO-d₆) δ 8.48 (d, J=6.9 Hz, 2H), 6.72 (bs, 4H), 3.95 (apparent quartet, J=5.1 Hz, 2H), 3.60 (apparent ABq of doublets; down-field group centered at 3.71, J=9.9, 5.1 Hz, 2H; up-field group centered at 3.48, J=9.9, 6.3 Hz, 2H). ¹³C NMR (75 MHz, CDCl3) δ 172.9 (s), 164.9 (s), 147.0 (s), 127.0 (s), 62.9 (d), 55.7 (t). LCMS (5-95% gradient acetonitrile in 0.1% TFA over 10 min), single peak retention time=1.45 min on 30 mm column, (M+H)⁺=373. UV/vis (100 μM in PBS) λ_abs=434 nm. Fluorescence λ_ex=449 nm, λ_em=559 nm.

EXAMPLE 4

3,6-bis(bis(2-methoxyethyl)amino)-N²,N²,N⁵,N⁵-tetrakis(2-methoxyethyl) pyrazine-2,5-dicarboxamide bis TFA salt

##STR00012##

Step 1. Synthesis of 3,6-dibromopyrazine-2,5-dicarboxylic acid

##STR00013##

3,6-Diaminopyrazine-2,5-dicarboxylic acid (499 mg, 2.52 mmol) was dissolved in 48% hydrobromic acid (10 mL) and cooled to 0° C. in an ice-salt bath. To this stirred mixture was added a solution of sodium nitrite (695 mg, 10.1 mmol) in water (10 mL) dropwise so that the temperature remains below 5° C. The resulting mixture was stirred for 3 h at 5-15° C., during which time the red mixture became a yellow solution. The yellow solution was poured into a solution of cupric bromide (2.23 g, 10.1 mmol) in water (100 mL) and the resulting mixture was stirred at room temperature. After an addition 3 h, the aqueous mixture was extracted with EtOAc (3×). The combined extracts were dried (Na₂SO₄), filtered and concentrated to afford 440 mg (54% yield) 3,6-dibromopyrazine-2,5-dicarboxylic acid as a pale yellow solid: ¹³C NMR (75 MHz, CDCl3) δ 164.3 (s), 148.8 (s), 134.9 (s). HPLC (5-95% gradient acetonitrile in 0.1% TFA over 10 min), single peak retention time=2.95 min on 250 mm column.

Step 2. Synthesis of 3-(Bis(2-methoxyethyl)amino)-6-bromo-N²,N²,N⁵,N⁵-tetrakis(2-methoxyethyl)pyrazine-2,5-dicarboxamide

##STR00014##

The product from step 1 (440 mg, 1.36 mmol) was dissolved in DMF (25 mL), treated with HOBt-H₂O (624 mg, 4.08 mmol), and EDC-HCl (786 mg, 4.10 mmol) and stirred for 30 min at room temperature. Bis(2-methoxylethyl)amine (620 mL, 559 mg, 4.20 mmol) was added and the resulting mixture was stirred at room temperature for 16 h and concentrated. The residue was partitioned with water and EtOAc. The EtOAc layer was separated and the aqueous was extracted again with EtOAc. The combined organic layers were washed with 0.5 N HCl, saturated sodium bicarbonate, and brine. The organic layer was dried (Na₂SO₄), filtered and concentrated to afford 214 mg of 3-(bis(2-methoxyethyl)amino)-6-bromo-N²,N²,N⁵,N⁵-tetrakis(2-methoxyethyl)pyrazine-2,5-dicarboxamide (26% yield) as a brown oil: LCMS (5-95% gradient acetonitrile in 0.1% TFA over 10 min), single peak retention time=3.85 min on 30 mm column, (M+H)⁺=608.

Step 3

To the product from step 2 (116 mg, 0.19 mmol) was added bis(2-methoxylethyl)amine (3.0 mL, 2.71 g, 20.3 mmol) and a “spatula tip” of Pd(PPh₃)₄. The resulting mixture was heated to 140° C. for 2 h. The reaction was cooled and concentrated. The residue was purified by flash chromatography (SiO₂, 10/1 CHCl₃-MeOH). The resulting material was purified again by reverse phase medium pressure chromatography (C18, 10-50% manual gradient acetonitrile in 0.1% TFA) to afford 12 mg (10% yield) of Example 4 as an orange-brown film: LCMS (15-95% gradient acetonitrile in 0.1% TFA over 10 min), single peak retention time=3.85 min on 250 mm column, (M+H)⁺=661. UV/vis (100 μM in PBS) λ_abs=434 nm. Fluorescence λ_ex=449 nm, λ_em=559 nm.

EXAMPLE 5

3,6-diamino-N²,N⁵-bis(2-aminoethyl)pyrazine-2,5-dicarboxamide bis TFA salt

##STR00015##

Step 1. Synthesis of 3,6-diamino-N²,N⁵-bis[2-(tert-butoxycarbonyl)aminoethyl]pyrazine-2,5-dicarboxamide

##STR00016##

A mixture of sodium 3,6-diaminopyrazine-2,5-dicarboxylate (500 mg, 2.07 mmol), tert-butyl 2-aminoethylcarbamate (673 mg, 4.20 mmol), HOBt-H₂O (836 mg, 5.46 mmol) and EDC-HCl (1.05 g, 5.48 mmol) in DMF (25 mL) was stirred for 16 h and concentrated. Work up as in Example 1 afforded 770 mg (76% yield) of the bisamide as an orange foam: ¹NMR (300 MHz, DMSO-d₆) major comformer, δ 8.44 (t, J=5.7 Hz, 2H), 6.90 (t, J=5.7 Hz, 2H), 6.48 (bs, 4H), 2.93-3.16 (complex m, 8H), 1.37 (s, 9H), 1.36 (s, 9H). ¹³C NMR (75 MHz, DMSO-d₆), conformational isomers δ 165.1 (s), 155.5 (bs), 155.4 (bs), 146.0 (s), 126.2 (s), 77.7 (bs), 77.5 (bs), 45.2 (bt), 44.5 (bt), 28.2 (q).

Step 2

To the product from step 1 (770 mg, 1.60 mmol) in methylene chloride (100 mL) was added TFA (25 mL) and the reaction was stirred at room temperature for 2 h. The mixture was concentrated and the residue taken up into methanol (15 mL). Ether (200 mL) was added and the orange solid precipitate was isolated by filtration and dried at high vacuum to afford 627 mg (77% yield) of Example 5 as an orange powder: ¹NMR (300 MHz, DMSO-d₆) δ 8.70 (t, J=6 Hz, 2H), 7.86 (bs, 6H), 6.50 (bs, 4H), 3.46-3.58 (m, 4H), 3.26-3.40 (m, 4H). ¹³C NMR (75 MHz, DMSO-d₆) δ 166.4 (s), 146.8 (s), 127.0 (s), 39.4 (t), 37.4 (t). LCMS (5-95% gradient acetonitrile in 0.1% TFA over 10 min), single peak retention time=3.62 min on 30 mm column, (M+H)⁺=283. UV/vis (100 μM in PBS) λ_abs=435 nm. Fluorescence (100 nM) λ_ex=449 nm, λ_em=562 nm.

EXAMPLE 6

3,6-Diamino-N²,N⁵-bis(D-Aspartate)-pyrazine-2,5-dicarboxamide

##STR00017##

Step 1. Synthesis of 3,6-Diamino-N²,N⁵-bis(benzyl D-Obenzyl-Aspartate)-pyrazine-2,5-dicarboxamide

##STR00018##

A mixture of sodium 3,6-diaminopyrazine-2,5-dicarboxylate (600 mg, 2.48 mmol), Asp(OBn)-OMe-p-TosH salt (2.43 g, 5.00 mmol), HOBt-H₂O (919 mg, 6.00 mmol) and EDC-HCl (1.14 g, 5.95 mmol) in DMF (50 mL) was treated with TEA (4 mL). The resulting mixture was stirred over night at room temperature. The reaction mixture was concentrated and the residue was partitioned with water and EtOAc. The EtOAc layer was separated and washed successively with saturated sodium bicarbonate, water and brine. The EtOAc solution was dried (Na₂SO₄), filtered and concentrated. The residue was purified by flash chromatography (SiO₂, 50/1 CHCl₃-MeOH to 10/1) to afford 1.15 g of the bis-amide (58% yield) as a yellow foam: ¹NMR (500 MHz, CDCl₃) δ 8.61 (d, J=8.4 Hz, 2H), 7.29-7.39 (m, 20H), 5.85 (bs, 4H), 5.22 (ABq, J=10.0 Hz, Δν=17.3 Hz, 4H), 5.10 (ABq, J=12.2 Hz, Δν=34.3 Hz, 4H), 5.06-5.09 (obs m, 2H), 3.11 (ABq of d, J=17.0, 5.14 Hz, Δν=77.9 Hz, 4H). ¹³C NMR (75 MHz, CDCl₃) δ 170.7 (s), 170.7 (s), 165.4 (s), 147.0 (s), 135.7 (s), 135.6 (s), 129.0 (d), 128.9 (d), 128.8 (d), 128.75 (d), 128.7 (d), 126.9 (s), 68.0 (t), 67.3 (t), 49.1 (d), 37.0 (t). LCMS (50-95% gradient acetonitrile in 0.1% TFA over 10 min), single peak retention time=5.97 min on 250 mm column, (M+H)⁺=789.

Step 2

To the product from step 1 (510 mg, 0.65 mmol) was added THF (20 mL) and water (10 mL). The this stirred mixture was added 10% Pd(C) (500 mg) and ammonium formate (1 g). The resulting mixture was heated to 60° C. for 2 h and allowed to cool to room temperature. The mixture was filtered through celite and concentrated. The resulting material was purified again by reverse phase medium pressure chromatography (C18, 10-70% manual gradient acetonitrile in 0.1% TFA) to afford 137.8 mg (54% yield) of Example 6 as an orange solid: ¹NMR (300 MHz, DMSO-d₆) δ 8.62 (d, J=8.4 Hz, 2H), 6.67 (bs, 4H), 4.725 (dt, J=8.4, 5.4 Hz, 2H), 2.74-2.88 (complex m, 4H). ¹³C NMR (75 MHz, DMSO-d₆) δ 172.6 (s), 165.2 (s), 147.0 (s), 126.6 (s), 60.8 (t), 49.1 (d). LCMS (5-95% gradient acetonitrile in 0.1% TFA over 10 min), single peak retention time=4.01 min on 250 mm column, (M+H)⁺=429. UV/vis (100 μM in PBS) λ_abs=433 nm. Fluorescence (100 nM) λ_ex=449 nm, λ_em=558 nm.

EXAMPLE 7

3,6-Diamino-N²,N⁵-bis(14-oxo-2,5,8,11-tetraoxa-15-azaheptadecan-17-yl) pyrazine-2,5-dicarboxamide

##STR00019##

To a solution of Example 5 (77.4 mg, 0.15 mmol) in DMF (5 mL) was added TEA (151 mg, 1.49 mmol) and 2,5-dioxopyrrolidin-1-yl 2,5,8,11-tetraoxatetradecan-14-oate (113 mg, 0.34 mmol) and the reaction was stirred for 16 h at room temperature. The reaction was concentrated and the residue was purified by medium pressure revered phase chromagraphy (LiChroprep RP-18 Lobar (B) 25×310 mm—EMD chemicals 40-63 μm, ˜70 g, 90/10 to 80/20 0.1% TFA-ACN) to afford 37.4 mg (35% yield) of example 7 as an orange film: ¹NMR (300 MHz, DMSO-d₆) δ 8.47 (t, J=5.7 Hz, 2H), 7.96 (t, J=5.4 Hz, 2H), 3.20-3.60 (complex m, 36H), 3.47 (s, 3H), 3.46 (s, 3H), 2.30 (t, J=6.3 Hz, 4H). ¹³C NMR (75 MHz, DMSO-d₆) δ 170.2 (s), 165.1 (s), 146.0 (s), 126.2 (s), 71.2 (t), 69.7 (t), 69.6 (t), 69.5 (t), 69.4 (t), 66.7 (t), 58.0 (q), 38.2 (t), 36.2 (t). LCMS (5-95% gradient acetonitrile in 0.1% TFA over 10 min), single peak retention time=4.01 min on 250 mm column, (M+H)⁺=719, (M+Na)⁺=741. UV/vis (100 μM in PBS) λ_abs=437 nm. Fluorescence (100 nM) λ_ex=437 nm, λ_em=559 nm.

EXAMPLE 8

3,6-Diamino-N²,N⁵-bis(26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-azanonacosan-29-yl)pyrazine-2,5dicarboxamide

##STR00020##

To a solution of Example 5 (50.3 mg, 0.10 mmol) in DMF (5 mL) was added TEA (109 mg, 1.08 mmol) and 2,5-dioxopyrrolidin-1-yl 2,5,8,11,14,17,20,23-octaoxahexacosan-26-oate (128 mg, 0.25 mmol) and the reaction was stirred for 16 h at room temperature. The reaction was concentrated and the residue was purified by medium pressure revered phase chromagraphy (LiChroprep RP-18 Lobar (B) 25×310 mm—EMD chemicals 40-63 μm, ˜70 g, 90/10 to 80/20 0.1% TFA-ACN) to afford 87.9 mg (82% yield) of example 8 as an orange film: ¹NMR (300 MHz, DMSO-d₆) δ 8.46 (t, J=5.7 Hz, 2H), 7.96 (t, J=5.4 Hz, 2H), 3.16-3.73 (complex m, 74H), 2.28-2.32 (m, 2H). ¹³C NMR (75 MHz, DMSO-d₆)—multiple conformations—δ 170.1 (s), 169.9 (s) 169.8 (s), 165.1 (s), 146.0 (s), 126.2 (s). 71.2 (t), 69.7 (t), 69.6 (t), 69.5 (t), 66.7 (t), 58.0 (q), 38.2 (t), 36.2 (t). LCMS (15-95% gradient acetonitrile in 0.1% TFA over 10 min), single peak retention time=5.90 min on 250 mm column, (M+H)⁺=1071, (M+2H)²⁺=536. UV/vis (100 μM in PBS) λ_abs=438 nm. Fluorescence (100 nM) λ_ex=438 nm, λ_em=560 nm.

EXAMPLE 9

3,6-Diamino-N²,N⁵-bis(38-oxo-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxa-39-azahentetracontan-41-yl)pyrazine-2,5-dicarboxamide

##STR00021##

To a solution of Example 5 (53.1 mg, 0.10 mmol) in DMF (5 mL) was added TEA (114 mg, 1.13 mmol) and 2,5-dioxopyrrolidin-1-yl 2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontan-38-oate (144 mg, 0.21 mmol) in DMF (2.0 mL) and the resulting mixture was stirred for 16 h thereafter. The reaction was concentrated and the residue was purified by medium pressure revered phase chromagraphy (LiChroprep RP-18 Lobar (B) 25×310 mm—EMD chemicals 40-63 μm, ˜70 g, 90/10 to 80/20 0.1% TFA-ACN) to afford 87.5 mg (61% yield) of example 9 as an orange film: ¹NMR (300 MHz, DMSO-d₆) δ 8.48 (t, J=5.7 Hz, 2H), 7.96 (t, J=5.4 Hz, 2H), 7.80-7.86 (m, 2H), 5.94 (bm, 2H), 3.30-3.60 (complex m, 106H), 2.26-2.33 (m, 4H). ¹³C NMR (75 MHz, DMSO-d₆) δ 170.2 (s), 165.1 (s), 146.0 (s), 126.2 (s), 71.2 (t), 69.7 (t), 69.6 (t), 69.5 (t), 66.7 (t), 58.0 (q), 38.2 (t), 36.2 (t). LCMS (15-95% gradient acetonitrile in 0.1% TFA over 10 min), single peak retention time=5.90 min on 250 mm column, (M+2H)²⁺=712. UV/vis (100 μM in PBS) λ_abs=449 nm. Fluorescence (100 nM) λ_ex=449 nm, λ_em=559 nm.

EXAMPLE 10

Bis(2-(PEG-5000)ethyl) 6-(2-(3,6-diamino-5-(2-aminoethylcarbamoyl)pyrazine-2-carboxamido)ethylamino)-6-oxohexane-1,5-diyldicarbamate

##STR00022##

A solution of Example 5 (25 mg, 0.049 mmol) in DMF (30 mL) was treated with TEA (1 mL) and m-PEG2-NHS (1 g, 0.1 mmol) and the resulting mixture was stirred for 48 h at room temperature. The mixture was concentrated and the residue was partially purified by gel filtration chromatography (G-25 resin, water). The product was concentrated and further purified by reverse phase medium pressure chromatography (C18, 10-70% manual gradient acetonitrile in 0.1% TFA) to afford 137.8 mg (54% yield) of Example 10 as a tan waxy solid: Maldi MS m/z=11393.

EXAMPLE 11

(R)-2-(6-(bis(2-methoxyethyl)amino)-5-cyano-3-morpholinopyrazine-2-carboxamido)succinic acid

##STR00023##

Step 1. Synthesis of 2-amino-5-bromo-3,6-dichloropyrazine

##STR00024##

A solution of 2-amino-6-chloropyrazine (25 g, 193.1 mmol) in MeOH (500 mL) was treated with NBS (34.3 g, 193.1 mmol), portion-wise, over 1 hour. The resulting mixture was stirred for 16 hours thereafter. TLC analysis at this time shows a small amount of starting material remaining. Another 1.4 g NBS added and reaction heated to 50° C. for 2 hours. The mixture was then cooled to 38° C. and treated with NCS (25.8 g, 193.1 mmol). The reaction mixture was heated to 50° C. for 16 hours thereafter. The mixture was then cooled to room temperature and treated with water (500 mL). The precipitate was collected by filtration and dried in a vacuum dessicator to afford 45.4 g (97% yield) of 2-amino-5-bromo-3,6-dichloropyrazine as a white solid: ¹³C NMR (75 MHz, CDCl₃) δ 149.9 (s), 145.6 (s), 129.6 (s), 121.5 (s). LCMS (15-95% gradient acetonitrile in 0.1% TFA over 10 min), single peak retention time=4.51 min on 30 mm column, (M+H)⁺=244, (M+H+ACN)⁺=285.

Step 2. Synthesis of 5-amino-3,6-dichloropyrazine-2-carbonitrile

##STR00025##

A mixture of CuCN (8.62 g, 96.3 mmol) and NaCN (4.72 g, 96.3 mmol) was heated under high vacuum to 90° C. The resulting mixture was subjected to three Argon/Vacuum cycles and placed under a final positive pressure of Argon. The mixture was allowed to cool to room temperature and DMF (150 mL) was added. The heterogenous mixture was heated to 130° C. for 2.5 hours. To the resulting homogeneous mixture of sodium dicyanocuprate was added a solution of the product from step 1 (15.6 g, 64.2 mmol) dissolved in DMF (150 mL), dropwise, over 1 hour. The temperature was gradually raised to 150° C. and the resulting mixture was stirred at this temperature for 10 hours thereafter. The reaction was then allowed to cool to room temperature and poured into water (1 L). The resulting mixture was extracted with EtOAc (3×) and the combined extracts were filtered to remove a flocculant dark solid, washed with brine, dried (Na₂SO₄), filtered again and concentrated. Purification by flash column chromatography (SiO₂, 10/1 hexanes-EtOAc to 3/1) to afford 6.70 g (55% yield) of the nitrile product as a tan solid: ¹³C NMR (75 MHz, CDCl₃) δ 153.9 (s), 149.1 (s), 131.7 (s), 115.4 (s), 111.0 (s). GCMS (Inj. temperature=280° C., 1.0 mL/min helium flow rate, temperature program: 100° C. (2 min hold), ramp to 300° C. @ 10° C./min (2 min hold), major peak retention time=16.556 min, m/z (EI)=188, 190.

Step 3. Synthesis of 5-amino-3-(bis(2-methoxyethyl)amino)-6-chloropyrazine-2-carbonitrile

##STR00026##

To the product from step 2 (1.00 g, 5.29 mmol) in ACN (20 mL) was added bis(2-methoxyethyl)amine (3.0 mL, 2.71 g, 20.3 mmol) and the reaction mixture was heated to 70° C. for 16 hours thereafter. The reaction was cooled and concentrated. The residue was partitioned with EtOAc and water. The organic layer was separated and the aqueous was extracted again with EtOAc. The combined organic extracts were washed with brine, dried (Na₂SO₄), filtered and concentrated. Purification by flash column chromatography (SiO₂, 10/1 hexanes-EtOAc to 1/1) afforded 950 mg (63% yield) of the desired adduct as a yellow solid: ¹NMR (300 MHz, CDCl₃) δ 7.47 (bs, 2H), 3.77 (t, J=5.7 Hz, 4H), 3.52 (t, J=5.4 Hz, 4H), 3.25 (s, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 154.7 (s), 152.0 (s), 120.9 (s), 119.5 (s), 95.8 (s), 71.0 (t), 59.1 (q), 50.0 (t). LCMS (50-95% gradient acetonitrile in 0.1% TFA over 10 min), single peak retention time=4.91 mm on 250 mm column, (M+H)⁺=286, (M+Na)⁺=308, (M+Na+ACN)⁺=349.

Step 4. Synthesis of 3-(bis(2-methoxyethyl)amino)-5-bromo-6-chloropyrazine-2-carbonitrile

##STR00027##

To the product from step 3 (1.39 g, 4.88 mmol) in 48% hydrobromic acid (20 mL) at 0° C. (ice-salt bath), was added a solution of sodium nitrite (673 mg, 9.75 mmol) in water (10 mL) dropwise over 30 min. The resulting mixture was stirred at 0-5° C. for 1 h and poured into a stirred solution of CuBr₂ (1.64 g, 7.34 mmol) in water (100 mL). The resulting mixture was stirred for 16 h at room temperature thereafter. The mixture was extracted with EtOAc (3×). The combined organic layers were dried (Na₂SO₄), filtered and concentrated. Purification by flash column chromatography (SiO₂, 50/1 CHCl3-MeOH) afforded 1.00 g (58% yield) of the bromide as a orange-brown solid: ¹NMR (300 MHz, CDCl₃) δ 3.99 (t, J=5.4 Hz, 4H), 3.64 (t, J=5.4 Hz, 4H), 3.35 (s, 6H). ¹³C NMR (75 MHz, CDCl₃) δ 152.8 (s), 140.8 (s), 133.4 (s), 117.2 (s), 108.3 (s), 70.4 (t), 59.1 (t), 50.5 (q). LCMS (50-95% gradient acetonitrile in 0.1% TFA over 10 min), single peak retention time=4.55 min on 250 mm column, (M+H)⁺=349, 351.

Step 5. Synthesis of 3-(bis(2-methoxyethyl)amino)-6-chloro-5-(furan-2-yl)pyrazine-2-carbonitrile

##STR00028##

A mixture of the product from step 4 (1.0 g, 2.87 mmol), 2-furanboronic acid (643 mg, 5.75 mmol), Cs₂CO₃ (3.31 g, 10.2 mmol), TFP (35 mol %, 236 mg, 1.02 mmol), and Pd₂dba₃-CHCl₃ (5 mol %, 10 mol % Pd, 150 mg) was subjected to 3 vacuum/Argon cycles and placed under a positive pressure of Argon. Anhydrous dioxane (50 mL) was added and the reaction mixture was heated to 75° C. for 16 h thereafter. The reaction mixture was cooled to room temperature, diluted with EtOAc (100 mL) and filtered through a medium frit. Concentration and purification of the residue by flash chromatography (SiO₂, 50/1 CHCl₃-MeOH) afforded the 757 mg of the furan adduct (78% yield) as a tan powder: LCMS (5-95% gradient acetonitrile in 0.1% TFA over 10 min), single peak retention time=6.41 min on 250 mm column, (M+H)⁺=337.

Step 6. Synthesis of 6-(bis(2-methoxyethyl)amino)-3-chloro-5-cyanobyrazine-2-carboxylic acid

##STR00029##

To a well stirred mixture of ACN (11 mL), CCl4 (7 mL), and water (11 mL) were added sodium periodate (1.07 g, 5.00 mmol) and RuO2.H₂O (13.3 mg, 0.10 mmol), sequentially. The resulting mixture was stirred vigorously at room temperature for 30 min and treated with sodium bicarbonate (2.10 g, 25.0 mmol) followed by water (5 mL). Vigorous stirring for another 15 minutes was followed by the addition of a solution of the product from Step 5 (276 mg, 0.82 mmol) dissolved in ACN (1 mL). The green mixture was stirred at room temperature for 5.5 h. The mixture was transferred to a separatory funnel and extracted with EtOAc. The aqueous layer was adjusted to pH˜3.5 and extracted again with EtOAc (2×). The combined extracts were washed with 20% sodium bisulfite and brine and dried (Na₂SO₄). Filtration and concentration afforded 140 mg (54% yield) of carboxylic acid as a pale yellow solid: LCMS (5-95% gradient acetonitrile in 0.1% TFA over 10 min), single peak retention time=5.05 min on 250 mm column, (M+H)⁺=315.

Step 7. Synthesis of (R)-dibenzyl 2-(6-(bis(2-methoxyethyl)amino)-3-chloro-5-cyanopyrazine-2-carboxamido)succinate

##STR00030##

A mixture of the product from step 6 (140 mg, 0.45 mmol), EDC.HCl (128 mg, 0.67 mmol) and HOBt.H₂O (102 mg, 0.67 mmol) in anhydrous DMF (25 mL) was stirred together at room temperature for 30 min. To this stirred mixture was added (R)-dibenzyl 2-aminosuccinate p-TsOH salt (213 mg, 0.44 mmol) followed by TEA (1 mL). The resulting mixture was stirred for 16 h thereafter. The reaction mixture was concentrated and partitioned with EtOAc and saturated sodium bicarbonate solution. The EtOAc layer was separated and washed with saturated sodium bicarbonate and brine, dried (Na₂SO₄), filtered and concentrated to afford 240 mg (88% yield) of the pyrazine amide as an orange foam: LCMS (15-95% gradient acetonitrile in 0.1% TFA over 10 min), single peak retention time=8.76 min on 250 mm column, (M+H)⁺=610, (M+Na)⁺=632.

Step 8. (R)-dibenzyl 2-(6-(bis(2-methoxyethyl)amino)-5-cyano-3-morpholinopyrazine-2-carboxamido)succinate

##STR00031##

To the product from step 7 (240 mg, 0.39 mmol) was added morpholine (5 mL). The reaction mixture was heated to 70° C. for 2 h. The mixture was cooled and concentrated. The residue was partitioned with EtOAc and water. The EtOAc layer was separated and washed with saturated sodium bicarbonate and brine. The EtOAc layer was dried (Na₂SO₄), filtered and concentrated. Purification by flash column chromatography (SiO₂, 3:1 to 1:1 hexanes-EtOAc) afforded 199 mg (75% yield) of the morpholine adduct as an orange foam: LCMS (15-95% gradient acetonitrile in 0.1% TFA over 10 min), single peak retention time=8.76 min on 250 mm column, (M+H)⁺=661, (M+Na)⁺=683.

Step 9. Synthesis of Example 11

##STR00032##

The dibenzyl ester (115 mg, 0.17 mmol) in THF (10 mL) was added 1.0 N sodium hydroxide (4 mL). The mixture was stirred for 1 h at room temperature. The pH was adjusted to ˜2 with 1.0 N HCl and the solution was concentrated. Purification of the residue by medium pressure reversed phase chromatography (LiChroprep RP-18 Lobar (B) 25×310 mm EMD chemicals 40-63 μm, ˜70 g, 90/10 to 50/50 0.1% TFA-ACN) afforded 32 mg (27% yield) of example 11 as an orange solid: LCMS (15-95% gradient acetonitrile in 0.1% TFA over 10 min), single peak retention time=4.47 min on 250 mm column, (M+H)⁺=481. UV/vis (100 μM in PBS) λ_abs=438 nm. Fluorescence (100 nM) λ_ex=449 nm, λ_em=570 nm.

EXAMPLE 12

Protocol for Assessing Renal Function

An example of an in vivo renal monitoring assembly 10 is shown in FIG. 2 and includes a light source 12 and a data processing system 14. The light source 12 generally includes or is interconnected with an appropriate device for exposing at least a portion of a patient's body to light therefrom. Examples of appropriate devices that may be interconnected with or be a part of the light source 12 include, but are not limited to, catheters, endoscopes, fiber optics, ear clips, hand bands, head bands, forehead sensors, surface coils, and finger probes. Indeed, any of a number of devices capable of emitting visible and/or near infrared light of the light source may be employed in the renal monitoring assembly 10.

Still referring to FIG. 2, the data processing system 14 of the renal monitoring assembly 10 may be any appropriate system capable of detecting spectral energy and processing data indicative of the spectral energy. For instance, the data processing system 14 may include one or more lenses (e.g., to direct and/or focus spectral energy), one or more filters (e.g., to filter out undesired wavelengths of spectral energy), a photodiode (e.g., to collect the spectral energy and convert the same into electrical signal indicative of the detected spectral energy), an amplifier (e.g., to amplify electrical signal from the photodiode), and a processing unit (e.g., to process the electrical signal from the photodiode). This data processing system 14 is preferably configured to manipulate collected spectral data and generate an intensity/time profile and/or a concentration/time curve indicative of renal clearance of a pyrazine derivative of the present invention from the patient 20. Indeed, the data processing system 14 may be configured to generate appropriate renal function data by comparing differences in manners in which normal and impaired cells remove the pyrazine derivative from the bloodstream, to determine a rate or an accumulation of the pyrazine derivative in organs or tissues of the patient 20, and/or to provide tomographic images of organs or tissues having the pyrazine derivative associated therewith.

In one protocol for determining renal function, an effective amount of a pyrazine derivative of the invention is administered to the patient (e.g., in the form for a pharmaceutically acceptable composition). At least a portion of the body of the patient 20 is exposed to visible and/or near infrared light from the light source 12 as indicated by arrow 16. For instance, the light from the light source 12 may be delivered via a fiber optic that is affixed to an ear of the patient 20. The patient may be exposed to the light from the light source 12 before or after administration of the pyrazine derivative to the patient 20. In some cases, it may be beneficial to generate a background or baseline reading of light being emitted from the body of the patient 20 (due to exposure to the light from the light source 12) before administering the pyrazine derivative to the patient 20. When the pyrazine derivative that is in the body of the patient 20 is exposed to the light from the light source 12, the pyrazine derivative emanates light (indicated by arrow 18) that is detected/collected by the data processing system 14. Initially, administration of the pyrazine derivative to the patient 20 generally enables an initial spectral signal indicative of the initial content of the pyrazine derivative in the patient 20. The spectral signal then tends to decay as a function of time as the pyrazine derivative is cleared from the patient 20. This decay in the spectral signal as a function of time is indicative of the patient's renal function. For example, in a first patient exhibiting healthy/normal renal function, the spectral signal may decay back to a baseline in a time of T. However, a spectral signal indicative of a second patient exhibiting deficient renal function may decay back to a baseline in a time of T+4 hours. As such, the patient 20 may be exposed to the light from the light source 12 for any amount of time appropriate for providing the desired renal function data. Likewise, the data processing system 14 may be allowed to collect/detect spectral energy for any amount of time appropriate for providing the desired renal function data.

REFERENCES

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Claims

1. A pharmaceutical composition comprising a compound of Formula I, wherein:

2. The pharmaceutical composition as recited in claim 1 further comprising a pharmaceutically acceptable excipient.

3. The pharmaceutical composition as recited in claim 2 wherein the pharmaceutically acceptable excipient is chosen from a diluent, preservative, solubilizer, emulsifier, adjuvant, and carrier.

4. The pharmaceutical composition as recited in claim 2 wherein the pharmaceutical acceptable excipient is phosphate buffered saline.

5. The pharmaceutical composition as recited in claim 2 wherein the pharmaceutical composition is a liquid.

6. The pharmaceutical composition as recited in claim 1, wherein at least one of X¹ and X² is —CONH(PS) or —CO(AA).

7. The pharmaceutical composition as recited in claim 1, wherein both X¹ and X² are —CO(AA).

8. The pharmaceutical composition as recited in claim 7, wherein each instance of AA is one or more of D-α-amino acids.

9. The pharmaceutical composition as recited in claim 8, wherein each instance of AA is a single D-α-amino acid.

10. The pharmaceutical composition as recited in claim 8, wherein AA is selected from the group consisting of D-aspartic acid, D-asparagine, D-arginine, D-histidine, D-lysine, D-glutamic acid, D-glutamine, D-serine, and D-homoserine.

11. The pharmaceutical composition as recited in claim 8, wherein AA is selected from the group consisting of D-serine and D-aspartic acid.

12. The pharmaceutical composition as recited in claim 8, wherein AA is D-serine.

13. The pharmaceutical composition as recited in claim 9, wherein AA is D-serine, Y¹ and Y² are each —NR⁴R⁵ with R⁴=R⁵=H.

14. The pharmaceutical composition as recited in claim 7, wherein each instance of AA is one or more of L-α-amino acids.

15. The pharmaceutical composition as recited in claim 14, wherein each instance of AA is a single L-α-amino acid.

16. The pharmaceutical composition as recited in claim 14, wherein AA is selected from the group consisting of L-aspartic acid, L-asparagine, L-arginine, L-histidine, L-lysine, L-glutamic acid, L-glutamine, L-serine, and L-homoserine.

17. The pharmaceutical composition as recited in claim 14, wherein AA is selected from the group consisting of L-serine and L-aspartic acid.

18. The pharmaceutical composition as recited in claim 14, wherein AA is L-serine.

19. The pharmaceutical composition as recited in claim 15, wherein AA is L-serine, Y¹ and Y² are each —NR⁴R⁵ with R⁴=R⁵=H.

20. The pharmaceutical composition as recited in claim 4, wherein X¹ and X² are —CO(AA), AA is a single D-α-amino acid that is D-serine, Y¹ and Y² are each —NR⁴R⁵ with R⁴=R⁵=H.

21. The pharmaceutical composition as recited in claim 4, wherein X¹ and X² are —CO(AA), AA is a single L-α-amino acid that is L-serine, Y¹ and Y² are each —NR⁴R⁵ with R⁴=R⁵=H.