Solid phase synthesis of NIR fluorescent probe

Patent metadata

Field	Value
Publication number	US-10822371-B2
Application number	US-201816769633-A
Country	US
Kind code	B2
Filing date	Dec 5, 2018
Priority date	Dec 6, 2017
Publication date	Nov 3, 2020
Grant date	Nov 3, 2020

Abstract

The present invention discloses a solid-phase process for the preparation of a Near Infra-Red (NIR) fluorescent probe characterized by an aza-bicycloalkane based cyclic peptide labelled with a Cy5.5 dye moiety and used in the guided surgery of tumors and pathologic regions.

First claim

The invention claimed is:

1. A process on solid support for the preparation of the cRGD-Cy5.5 conjugate of formula (I)

which comprises the steps of:
a) synthesising a tripeptide of formula (II) on a solid support

 wherein P 1 and P 2 are protecting groups and S is the solid support;
b) coupling of the tripeptide of formula (II) linked to the solid support with an aza-bicycloalkane of formula (III)

 wherein P 3 is a protecting group, to provide a compound of formula (IV)

wherein P 1 , P 2 , P 3 and S are as defined above;
c) removing the protecting groups P 2 and P 3 from compound (IV) to provide a compound of formula (V)

 wherein P 1 and S are as defined above;
d) performing an intramolecular cyclization to provide a compound of formula (VI)

 wherein P 1 and S are as defined above;
e) subjecting the resulting compound of formula (VI) to a reduction reaction to provide a corresponding amino-derivative of formula (VII)

 wherein P 1 and S are as defined above;
f) conjugating of the resulting compound of formula (VII) with a compound of formula (VIII)

 to obtain a compound of formula (IX)

 wherein P 1 and S are ad defined above;
g) removing the protecting group P 1 and cleavaging the peptido-like compound (IX) from the solid support S to obtain the compound of formula (I) as defined above.

2. The process according to claim 1 , further comprising the steps of
h) purification of the compound of formula (I) obtained in step g), and optionally
i) lyophilization of the final product.

3. The process according to claim 1 , wherein P 1 is a protecting group selected from 2,2,4,6,7-pentamethyl-2,3-dihydro-1-benzofuran-5-sulfonyl (Pbf), 2,3,6-trimethyl-4-methoxybenzene-sulfonyl (Mtr), p-toluenesulfonyl (Tos), trifluoroacetyl o-nitrobenzenesulfonyl (oNBS), 2,4-dinitrobenzenesulfonyl (dNBS), benzothiazole-2-sulfonyl (Bts), 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc) 2,3,6-trimethyl-4-methoxybenzene-sulfonyl (Mtr), p-toluenesulfonyl (Tos), trifluoroacetyl and tert-butyloxycarbonyl (Boc); P 2 is a protecting group selected from allyl, alkyl, t-butyl, trityl (Trt), 2,4-dimethoxybenzyl (DMB), benzyl (Bn), 9-fluorenylmethyl (Fm), 2-chlorotrityl (2-Cl-Trt), 2-phenylisopropyl (2-PhiPr), 5-phenyl-3,4-ethylenedioxythenyl derivatives (Phenyl-EDOTn), 4-(N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]-amino)benzyl (Dmab), carbamoylmethyl (Cam), p-nitrobenzyl (pNB), 2-trimethylsilylethyl (TMSE), (2-phenyl-2-trimethylsiylyl)ethyl (PTMSE), 2-(trimethylsilyl)isopropyl (Tmsi), 2,2,2-trichloroethyl (Tce), p-hydroxyphenacyl, 4,5-dimethoxy-2-nitrobenzyl (Dmnb), 1,1-dimethylallyl (Dma), pentaamine cobalt (III), β-menthyl (Men), β-3-methylpent-3-yl (Mpe) and 4-(3,6,9-trioxadecyl)oxybenzyl (TEGBz or TEGBn); and P 3 is a protecting group selected from allyloxycarbonyl (alloc), benzyloxycarbonyl (Cbz), 2-(4-biphenyl)isopropyloxycarbonyl (Bpoc), 3,5-dimethoxyphenylisopropyloxycarbonyl (Ddz), 2-(4-nitrophenylsulfonyl)ethoxycarbonyl (Nsc), 1,1-dioxobenzo[b]thiophene-2-ylmethyloxycarbonyl (Bsmoc), (1,1-dioxonaphtho[1,2-b]thiophene-2-yl)methyloxycarbonyl (α-Nsmoc), 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde), tetrachlorophthaloyl (TCP), 2-phenyl(methyl)sulfonio)ethyloxycarbonyltetrafluoroborate (Pms), ethanesulfonylethoxycarbonyl (Esc), 2-(4-sulfophenylsulfonyl)ethoxycarbonyl (Sps), trichloroethyloxycarbonyl (Troc), p-nitrobenzyloxycarbonyl (pNZ), propargyloxycarbonyl (Poc), o-nitrobenzyloxycarbonyl (oNZ), 4-nitroveratryloxycarbonyl (NVOC), (2-nitrophenyl)propyloxycarbonyl (NPPOC), azidomethyloxycarbonyl (Azoc) and trifluoroacetyl.

4. The process according to claim 3 , wherein P 1 is 2,2,4,6,7-pentamethyl-2,3-dihydro-1-benzofuran-5-sulfonyl (Pbf), P 2 is allyl and P 3 is allyloxycarbonyl (Alloc).

5. The process according to claim 1 , wherein the steps b), d) and f) are carried out in the presence of a coupling reagent and a base.

6. The process according to claim 5 , wherein the coupling reagent is selected from the group consisting of N,N,N′,N′-tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate (TBTU), N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU), N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate (HATU), O-(1H-6-Chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), 1-[1-(cyano-2-ethoxy-2-oxoethylidene-aminooxy)-dimethylamino-morpholino]-uronium hexafluorophosphate (COMU) and fluoro-N,N,N′,N′-tetramethylformamidinium hexafluorophosphate (TFFH) and the base is a tertiary amine.

7. The process according to claim 6 , wherein in step b) the coupling reagent is 1-[1-(cyano-2-ethoxy-2-oxoethylidene-aminooxy)-dimethylamino-morpholino]-uronium hexafluorophosphate (COMU) and the base is N,N-diisopropylethylamine (DIPEA).

8. The process according to claim 6 , wherein in step d) the coupling reagent is 7-azabenzotriazol-1-yloxy-tripyrrolidino-phosphonium hexafluorophosphate (PyAOP) and the base is N,N-diisopropylethylamine (DIPEA) and the ratio among the intermediate of formula (V), the coupling reagent and the base is about 1:1.5:3 (mol/mol).

9. The process according to claim 5 , wherein the ratio between the intermediate of formula (II) and coupling reagent is about 1:2 (mol/mol); the ratio between the intermediate of formula (III) and the coupling reagent is about 1:0.98 (mol/mol); and the ratio between the intermediate of formula (III) and the N,N-diisopropylethylamine is about 1:2 (mol/mol).

10. The process according to claim 1 , wherein the step c) is performed under Argon atmosphere with a prior activation phase of 3 min in the presence of an organosilane compound and a catalyst.

11. The process according to claim 10 , wherein the organosilane is phenylsilane (PhSiH 3 ) and the catalyst is tetrakis(triphenylphosphine)palladium (0) (Pd(PPH 3 ) 4 ), and the ratio between the intermediate (IV) and the phenylsilane is about 1:25 (mol/mol).

12. The process according to claim 1 , wherein step e) is carried out between 20° C. and 70° C., in the presence of an organophosphorous compound and water, for a period from 5 to 20 hours.

13. The process according to claim 1 , wherein step f) is performed by prior activation of the intermediate of formula (VIII).

14. An intermediate compound of formula (III)

wherein P 3 is a protecting group.

15. The process according to claim 2 , wherein P 1 is a protecting group selected from 2,2,4,6,7-pentamethyl-2,3-dihydro-1-benzofur

Assignees

• Bracco Imaging Spa

Inventors

• Campanella Raphael
• Clouet Anthony
• Lattuada Luciano

Classifications

• C07D471/04 Primary

  Ortho-condensed systems · CPC title

• C07K1/061 Primary

  using protecting groups · CPC title

• C07K1/10

  using coupling agents {(C07K1/006 takes precedence)} · CPC title