IP Library › Granted Patent US 9,513,294

Granted Patent B2

US 9,513,294 · App. 14/648,309 · Granted Dec 6, 2016

Megastokes amino-triazolyl-BODIPY compounds and applications to live neuron staining and human serum albumin FA1 drug site probing

Inventors: Young-Tae Chang (Singapore, SG); Jun Cheng Er (Singapore, SG); Cheryl Kit Mun Leong (Singapore, SG); Seong Wook Yun (Singapore, SG); Marc Vendrell Escobar (Edinburgh, GB); Mui Kee Tang (Singapore, SG)

Assignees: National University of Singapore; Agency for Science, Technology and Research

G01N33/582 A61K49/0021 C07F5/022 C09B23/0008 C09B23/04 C09B57/00 G01N21/6428 G01N33/5005 G01N33/5058 G01N2333/765 G01N2458/30

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Quick Facts

Patent No.: US 9,513,294
App. No.: 14/648,309
Granted: Dec 6, 2016
Kind: B2

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USPTO Patent Center↗ Full Patent PDF↗

Abstract

A library of novel amino-triazolyl-BODIPY compounds is described. Particular compounds of the library serve as selective fluorescent probes for human serum albumin (HSA) and for live primary neurons. The fluorescent probe for HSA binds uniquely and specifically to the fatty acid site 1 of HSA, and thus proves a valuable and unique probe for drugs that bind to such a site on HSA. Methods of synthesis for the library compounds are also described.

Claims (81)

1. A compound having the structure of Formula (I) or a pharmaceutically acceptable salt thereof:

wherein:

R 1 is H or CO(C 1 -C 6 )alkyl, wherein CO(C 1 -C 6 )alkyl is optionally substituted at any position with 1-3 substituents selected from halogen, hydroxyl, O-acetyl, NH-acetyl, or —NH 2 ;

R 2 and R 3 are each, independently, H, (C 1 -C 10 )alkyl, (C 6 -C 10 )aryl(C 0 -C 6 )alkyl, (C 3 -C 10 )heteroaryl, (C 3 -C 8 )cycloalkyl, (C 2 -C 10 )alkynyl or (C 2 -C 10 )alkenyl;

wherein (C 1 -C 10 )alkyl, (C 6 -C 10 )aryl(C 0 -C 6 )alkyl, (C 3 -C 10 )heteroaryl, (C 3 -C 8 )cycloalkyl, (C 2 -C 10 )alkynyl and (C 2 -C 10 )alkenyl are optionally substituted at any position with 1-5 substituents selected from (C 1 -C 6 )alkyl, (C 1 -C 6 )alkoxy, NH 2 , —NH(C 1 -C 6 alkyl), —NH(CO)(C 1 -C 6 alkyl), —NH(CO)(C 1 -C 6 haloalkyl), —NH(CO)(C 1 -C 6 alkoxy), —NH(CO)(C 1 -C 6 haloalkoxy), —NH(CO)(C 2 -C 6 alkenyl), —Si(C 1 -C 6 alkyl), —SO 2 (C 1 -C 6 alkyl), —SH, —B(OH) 2 , —OTosyl, —CO(C 1 -C 6 alkoxyl), —COH, —COOH, —CO(C 1 -C 6 alkyl) or halogen;

or R 2 and R 3 are taken together to form a ring, wherein the ring is optionally substituted with 1-5 additional substituents selected from halogen or (C 1 -C 6 )alkyl; and

R 4 is (C 1 -C 10 )alkyl, (C 3 -C 8 )cycloalkyl, (C 6 -C 10 )aryl, (C 2 -C 10 )alkenyl, (C 2 -C 10 )alkynyl, or (C 3 -C 10 )heteroaryl, further wherein R 4 is optionally substituted at any position with 1-5 substituents selected from (C 1 -C 10 )alkyl, (C 2 -C 10 )alkenyl, (C 2 -C 10 )alkynyl, (C 3 -C 8 )cycloalkyl, (C 1 -C 6 )alkoxy, (C 6 -C 10 )aryloxy, (C 6 -C 10 )aryl, amino, —NH(CO)(C 1 -C 6 alkyl), —NH(CO)(C 2 -C 6 alkenyl), —NH(CO)(C 2 -C 6 alkynyl), —NH(CO)(C 1 -C 6 haloalkyl), halo, OCF 3 , CF 3 , hydroxyl, or a halogen radioisotope.

2. The compound of claim 1 , wherein R 1 is H, COCH 3 , COCH 2 Cl, COCH 2 OH, COCH 2 NH 2 , or COCH 2 O(CO)CH 3 .

3. The compound of claim 2 , having the structure of Formula (II):

or a pharmaceutically acceptable salt thereof.

4. The compound of claim 2 , having the structure of Formula (III):

or a pharmaceutically acceptable salt thereof.

5. The compound of claim 2 , having the structure of Formula (IV):

or a pharmaceutically acceptable salt thereof.

6. The compound of claim 2 , having the structure of Formula (V):

or a pharmaceutically acceptable salt thereof.

7. The compound of claim 1 , wherein the compound is conjugated to a chemical reactive moiety selected from biotin, diazirine, acryloyl, haloacetyl, diaaziridine, N-hydroxysuccinimide ester, imidoester, pentafluorophenyl ester, hydroxymethyl phosphine, maleimide, pyridyldisulfide, thiosulfonate, vinylsulfone, hydrazide, alkoxyamine, aryl azide, benzophenone, isocyanate, alkyne or ketone.

8. A method for a solid phase synthesis of a compound of Formula (I) or a pharmaceutically acceptable salt thereof:

wherein:

R 1 is H or CO(C 1 -C 6 )alkyl, wherein CO(C 1 -C 6 )alkyl is optionally substituted at any position with 1-3 substituents selected from halogen, hydroxyl, O-acetyl, NH-acetyl, or —NH 2 ;

R 2 and R 3 are each, independently, H, (C 1 -C 10 )alkyl, (C 6 -C 10 )aryl(C 0 -C 6 )alkyl, (C 3 -C 10 )heteroaryl, (C 3 -C 8 )cycloalkyl, (C 2 -C 10 )alkynyl or (C 2 -C 10 )alkenyl;

or R 2 and R 3 are taken together to form a ring, wherein the ring is optionally substituted with 1-5 additional substituents selected from halogen or (C 1 -C 6 )alkyl; and

the method comprising:

(a) reacting a compound of Formula (VI):

with an azide under conditions sufficient to form a bis(azido)BODIPY compound of formula (VII):

(b) reacting the compound of Formula (VII) with an amine of the formula R 2 R 3 NH under conditions sufficient to form a compound of Formula (VIII):

(c) reacting the compound of Formula (VIII) with an alkyne of the formula R 4 —C≡CH in the presence of a copper (I) catalyst under conditions sufficient to form a compound of Formula (IX):

(d) removing the solid support resin from the compound of Formula (IX) to form an unbound BODIPY compound where R 1 in Formula (I) is H; and

(e) optionally reacting the unbound BODIPY compound of step d) with an acid chloride of the formula R 1 COCl to form a compound of Formula (I) where R 1 is CO(C 1 -C 6 )alkyl optionally substituted with 1-3 substituents selected from halogen, O-acetyl, or N-acetyl.

9. The method of claim 8 , wherein when step (e) is performed, the method comprises an additional step of treating the reaction mixture with a base under conditions sufficient to saponify an ester or amide linkage, wherein R 1 is CO(C 1 -C 6 )alkyl optionally substituted with 1-3 hydroxyl or —NH 2 substituents.

10. A method for a solution phase synthesis of a compound of Formula (I) or a pharmaceutically acceptable salt thereof:

wherein:

R 1 is H or CO(C 1 -C 6 )alkyl, wherein CO(C 1 -C 6 )alkyl is optionally substituted at any position with 1-3 substituents selected from halogen, hydroxyl, O-acetyl, NH-acetyl, or —NH 2 ;

R 2 and R 3 are each, independently, H, (C 1 -C 10 )alkyl, (C 6 -C 10 )aryl(C 0 -C 6 )alkyl, (C 3 -C 10 )heteroaryl, (C 3 -C 8 )cycloalkyl, (C 2 -C 10 )alkynyl or (C 2 -C 10 )alkenyl;

or R 2 and R 3 are taken together to form a ring, wherein the ring is optionally substituted with 1-5 additional substituents selected from halogen or (C 1 -C 6 )alkyl; and

the method comprising:

(a) reacting a compound of Formula (X):

with an azide under conditions sufficient to form a bis(azido)BODIPY compound of formula (XI):

(b) reacting the compound of Formula (XI) with an amine of the formula R 2 R 3 NH under conditions sufficient to form a compound of Formula (XII):

(c) reacting the compound of Formula (XII) with an alkyne of the formula R 4 —C≡CH in the presence of a copper (I) catalyst under conditions sufficient to form a compound of Formula (XIII):

(d) reducing the NO 2 group in the compound of Formula (XIII) to form a BODIPY compound where R 1 in Formula (I) is H; and

(e) optionally reacting the BODIPY compound of step d) with an acid chloride of the formula R 1 COCl to form a compound of Formula (I) where R 1 is CO(C 1 -C 6 )alkyl optionally substituted with 1-3 substituents selected from halogen, O-acetyl, or N-acetyl.

11. The method of claim 10 , wherein when step (e) is performed, the method comprises an additional step of treating the reaction mixture with a base under conditions sufficient to saponify an ester or amide linkage, wherein R 1 is CO(C 1 -C 6 )alkyl optionally substituted with 1-3 hydroxyl or —NH 2 substituents.

12. A method for detecting human serum albumin (HSA) in a sample of a biological fluid, comprising:

a) contacting a sample of biological fluid thought to contain HSA with the compound of claim 1 represented by Formula (I)or a pharmaceutically acceptable salt thereof:

wherein:

R 1 is H or CO(C 1 -C 6 )alkyl, wherein CO(C 1 -C 6 )alkyl is optionally substituted at any position with 1-3 substituents selected from halogen, hydroxyl, O-acetyl, NH-acetyl, or —NH 2 ;

R 2 and R 3 are each, independently, H, (C 1 -C 10 )alkyl, (C 6 -C 10 )aryl(C 0 -C 6 )alkyl, (C 3 -C 10 )heteroaryl, (C 3 -C 8 )cycloalkyl, (C 2 -C 10 )alkynyl or (C 2 -C 10 )alkenyl;

or R 2 and R 3 are taken together to form a ring, wherein the ring is optionally substituted with 1-5 additional substituents selected from halogen or (C 1 -C 6 )alkyl; and

to form an incubation media;

b) incubating the incubation media of step a) under conditions sufficient to form an incubated mixture; and

c) analyzing the mixture of step b) by fluorescence microscopy, wherein an increase in the fluorescence signal of the mixture relative to the fluorescence signal of the compound of Formula (I) not in the presence of a sample containing HSA is indicative of the presence of HSA.

13. The method of claim 12 , wherein a measure of intensity of the fluorescence signal is proportional to the concentration of the HSA present in the sample.

14. The method of claim 12 , wherein the compound of Formula (I) has the structure of formula (II):

or a pharmaceutically acceptable salt thereof.

15. A method for visualizing live neurons in a cell culture, comprising:

a) contacting a cell culture with the compound of claim 1 represented by Formula (I) or a pharmaceutically acceptable salt thereof:

wherein:

R 1 is H or CO(C 1 -C 6 )alkyl, wherein CO(C 1 -C 6 )alkyl is optionally substituted at any position with 1-3 substituents selected from halogen, hydroxyl, O-acetyl, NH-acetyl, or —NH 2 ;

R 2 and R 3 are each, independently, H, (C 1 -C 10 )alkyl, (C 6 -C 10 )aryl(C 0 -C 6 )alkyl, (C 3 -C 10 )heteroaryl, (C 3 -C 8 )cycloalkyl, (C 2 -C 10 )alkynyl or (C 2 -C 10 )alkenyl;

or R 2 and R 3 are taken together to form a ring, wherein the ring is optionally substituted with 1-5 additional substituents selected from halogen or (C 1 -C 6 )alkyl; and

to form an incubation media;

b) incubating the incubation media of step a) under conditions sufficient to stain the live neurons; and

c) visualizing the stained live neurons of step b) with fluorescence microscopy.

16. The method of claim 15 , wherein the compound of Formula (I) is a compound having the structure of Formula (XIV) or a pharmaceutically acceptable salt thereof:

wherein:

R 5 is present at any position with 1-5 substituents selected from H, (C 1 -C 6 )alkyl, (C 1 -C 6 )alkoxy, NH 2 , —NH(C 1 -C 6 alkyl), —NH(CO)(C 1 -C 6 alkyl), —NH(CO)(C 1 -C 6 haloalkyl), —NH(CO)(C 1 -C 6 alkoxy), —NH(CO)(C 1 -C 6 haloalkoxy), —NH(CO)(C 2 -C 6 alkenyl), —Si(C 1 -C 6 alkyl), —SO 2 (C 1 -C 6 alkyl), —SH, —B(OH) 2 , —OTosyl, —CO(C 1 -C 6 alkoxyl), —COH, —COOH, —CO(C 1 -C 6 alkyl) or halogen.

17. The method of claim 16 , wherein the compound of Formula (I) has the structure of Formula (V):

or a pharmaceutically acceptable salt thereof.

18. The compound of claim 1 , having the structure of Formula (XVI):

or a pharmaceutically acceptable salt thereof.

19. The compound of claim 1 , wherein the halogen radioisotope is 18 F.

Assignments (1)

ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded May 29, 2015

From: CHANG, YOUNG-TAE; ER, JUN CHENG; LEONG, CHERYL KIT MUN; YUN, SEONG-WOOK; ESCOBAR, MARC VENDRELL; TANG, MUI KEE

To: NATIONAL UNIVERSITY OF SINGAPORE; AGENCY FOR SCIENCE, TECHNOLOGY AND RESEARCH

Reel/Frame 035743/0068 →

Continuity (2)

Provisional Application 61745916 · Dec 26, 2012

Related Publication 20150309040A1 · Oct 29, 2015