Methods of Manufacture and Synthesis of Amino Acid Linking Groups Conjugated To Compounds Used For Targeted Imaging of Tumors

A method of synthesizing a conjugate having the formula

• or a pharmaceutically acceptable salt or isotope thereof, wherein:
• X is an amino acid or a derivative thereof, and
• Y is a dye that has a fluorescence excitation and emission spectra in the near infra red (NIR) range, and the compound maintains or enhances the fluorescence of the dye, comprising the steps of:
• a. mixing a pteroyl derivative compound and X in the presence of (-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU), a base such as Hunig's base (DIPEA) and a polar solvent to form pteroyl-X(OtBu);
• b. precipitating the pteroyl-X (OtBu) in the polar solvent with water or 1N HCl;
• c. adding a resulting product to TFA:TIPS:H2O (95:2.5:2.5) solvent to deprotect tBu group to form a pteroyl-X ligand;
• d. adding the pteroyl-X in TFA:TIPS:H2O solvent as a steady stream to a solution of ether such as methyl tertiary-butyl ether (MTBE) or diethyl ether to precipitate the ligand;
• e. filtering and washing the precipitated ligand with methyl tertiary-butyl ether (MTBE);
• f. drying the ligand under high vacuum conditions;
• g. dissolving the ligand in a polar solvent;
• h. adding a base to adjust the pH wherein the base is an inorganic base selected from the group consisting of sodium hydroxide (NaOH), potassium hydroxide (KOH), and potassium carbonate (K2CO3) or wherein the base is an organic base sodium tertiary-butoxide (NaOC(CH3));
• i. adding the solution of ligand in water or DMSO to Y in a water or DMSO and reflux to 70° C.-100° C. for 10-60 minutes;
• j. cooling the resulting mixture to room temperature;
• k. adding the resulting mixture to a stirred acetone to give a precipitate ligand-amino acid-fluorescent dye conjugate;
• and
• l. filtering the ligand-amino acid-fluorescent dye conjugate under high vacuum conditions.

A method of synthesizing a conjugate having the formula:

• where W, X, Y, Z each are H, Na, K+ or NH4+,
• comprising the steps of:
• a. mixing pteroic acid and amino acid in the presence of (-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU), Hunig's base (DIPEA) and a dimethylformamide (DMF);
• b. precipitating Pte_TFA_L_Tyr(OtBu)-OtBu in DMF with 1N HCl;
• c. Dissolving Pte_TFA_L_Tyr(OtBu)-OtBu precipitate in a TFA:TIPS:H2O (95:2.5:2.5) solvent to form Pte-TFA-L-Tyr solution;
• d. adding the Pte-TFA-L-Tyr solution as a steady stream to methyl tertiary-butyl ether (MTBE) to precipitate Pte_TFA_L_Tyr;
• e. filtering and washing Pte_TFA_L_Tyr precipitate with methyl tertiary-butyl ether (MTBE);
• f. drying the Pte_TFA_L_Tyr under high vacuum conditions;
• g. dissolving the resulting Pte_TFA_L_Tyr in water or DMSO;
• h. adding aqueous sodium hydroxide (NaOH) to adjust the pH and to deprotect TFA group;
• i. adding the solution of Pte_TFA_L_Tyr in water to S0456 fluorescent dye in water or DMSO and reflux at 70° C.-100° C. for 10 minutes;
• j. cooling the resulting mixture to room temperature;
• k. adding the resulting mixture to a stirred acetone to obtain a Pteroyl-Tyr-S0456 precipitate;
• and
• l. drying the Pteroyl-Tyr-S0456 conjugate with acetone under high vacuum conditions.

A method of synthesizing a conjugate in a solid phase having the formula:

• where W, X, Y, Z each are H, Na, K+ or NH4+,
• comprising the steps of:
• a. swelling a Fmoc-L-Tyr(OtBu)-Wang Resin with dichloromethane (DCM) and dimethylformamide (DMF) respectively in a solid phase peptide synthesis vessel;
• b. deprotecting the Fmoc group using 20% piperidine/DMF;
• c. washing the resin with dimethylformamide (DMF) and isopropyl alcohol (IPA);
• d. swelling the resin with dichloromethane (DCM);
• e. adding a solution of N10-(Trifluoroacetyl)pteroic acid in the presence of (-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU), Hunig's base (DIPEA) and dimethylformamide (DMF) to the resin;
• f. filtering the excess unreacted Pteroyl-N10-TFA in the presence of (-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU), Hunig's base (DIPEA) and dimethylformamide (DMF) from the resin;
• g. washing the resin with dimethylformamide (DMF) and isopropyl alcohol (IPA);
• h. swelling the resin with dichloromethane (DCM);
• i. drying the resin under argon;
• j. cleaving a resulting Pteroyl-TFA_L-Tyr compound from the resin with TFA:H2O:TIPS (95:2.5:2.5) to a ether solution such as methyl tertiary-butyl ether (MTBE) to precipitate of Pteroyl-TFA_L-Tyr;
• k. drying the Pte_TFA_L_Tyr under high vacuum conditions;
• l. dissolving the resulting Pte_TFA_L_Tyr in water or DMSO;
• m. adding aqueous sodium hydroxide (NaOH) to adjust the pH and to deprotect the TFA group;
• n. adding the solution of Pte_TFA_L_Tyr in water to S0456 fluorescent dye in water or DMSO and reflux at 70° C.-100° C. for 10 minutes;
• o. cooling the resulting mixture to room temperature;
• p. adding the resulting mixture to a stirred acetone to obtain a solid Pteroyl-Tyr-S0456 compound;
• and
• q. drying the Pteroyl-Tyr-S0456 under high vacuum conditions.

A method of synthesizing a conjugate having the formula:

• where W, X, Y, and Z are each H, Na, or NH4+,
• wherein C′ is any isotope of carbon, H′ is any isotope of hydrogen comprising the steps of:
• a. Adding a solution of N10-TFA-pteroic acid in the presence of (-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU), Hunig's base (DIPEA) and a dimethylformamide (DMF) to a solution of Tyr′ in dimethylformamide (DMF);
• b. adding the solution as a steady stream to aqueous 1N HCl to precipitate an intermediate compound;
• c. filtering and washing the intermediate precipitate with water under aspirator vacuum;
• d. drying the precipitated intermediate under high vacuum conditions;
• e. adding the resulting precipitate to a TFA:H2O:TIPS (95:2.5:2.5) solution;
• f. adding the solution as a steady stream to diethyl ether or MTBE to precipitate a pteroic acid-TFA-Tyr′ compound;
• g. filtering and washing the Pteroyl-TFA-Tyr′-precipitate with diethyl ether or MTBE under aspirator vacuum conditions;
• h. drying the Pteroyl-TFA-Tyr′-precipitate under high vacuum conditions;
• i. dissolving the Pteroyl-TFA-Tyr′ in water;
• j. adding an aqueous sodium hydroxide (NaOH) to adjust the pH;
• k. adding the aqueous Pteroyl-TFA-Tyr′-to a solution of S0456 fluorescent dye in water and refluxing at 80° C.-100° C. for 15 minutes;
• l. cooling the resulting mixture to room temperature;
• m. adding the resulting mixture to a stirred acetone to obtain an analog of Pteroyl-Tyr′-S0456 compound;
• and
• n. drying the analog of Pteroyl-Tyr′-S0456 conjugate with acetone under high vacuum.