Fluorescent probes

Patent metadata

Field	Value
Publication number	US-2024279710-A1
Application number	US-202218568591-A
Country	US
Kind code	A1
Filing date	Jun 23, 2022
Priority date	Jun 24, 2021
Publication date	Aug 22, 2024
Grant date	—

Abstract

A fluorescent probe that detects calpain enzyme activity with high sensitivity, wherein, enzymatic reactivity with a calpain is improved by bonding an amide group having α-carbon, which is bonded to an oxygen atom, to the N-terminal of a peptide chain. A fluorescent probe detects calpain activity with higher sensitivity because the probe is improved in reactivity with a calpain as compared to a HMRG fluorescent probe that has heretofore been reported. Specifically, the fluorescent probe relates to a compound represented by the following general formula (I) or a salt thereof.

First claim

1 . A compound represented by the following general formula (I) or a salt thereof:

where:
A and B each independently represent an amino acid residue, and the amino acid residues may be identical to or different from each other,
where a bond between A and NH bonded to A is an amide bond between a C-terminal of A and the NH, A and B are bonded through an amide bond, and a bond between B and a carbonyl group (C═O) bonded to B is an amide bond between an N-terminal of B and the carbonyl group (C═O);

R 1 s may be identical to or different from each other, and each represent a substituent bonded to a benzene ring;
“p” represents an integer of from 0 to 4;
R 2 , R 3 , R 4 , R 5 , R 6 , and R 7 each independently represent a hydrogen atom, a hydroxyl group (OH group), a halogen atom, or a linear, branched, or cyclic alkyl group that may be substituted;
R 8 and R 9 each independently represent a hydrogen atom, or a linear, branched, or cyclic alkyl group that may be substituted, or R 8 and R 9 may be bonded to form a ring, or R 3 and R 8 may be bonded to form a ring, and/or R 4 and R 9 may be bonded to form a ring;
X represents a linear or branched C1 to C3 alkylene group;
R 10 and R 11 each independently represent a hydrogen atom, a hydroxyl group (OH), an alkoxy group, or a linear, branched, or cyclic alkyl group that may be substituted, or R 10 and R 11 may be bonded to form a ring;
R 12 represents a hydrogen atom, a linear, branched, or cyclic alkyl group that may be substituted, a linear or branched alkenyl group that may be substituted, a linear or branched alkynyl group that may be substituted, an aryl group that may be substituted, or a heterocyclic group that may be substituted, or R 2 may be bonded to R 10 or R 11 to form a ring; and
L represents an amide bond, an ester bond, or -(linear or branched alkylene-O—) n — where “n” represents an integer of from 1 to 10, or L represents a single bond.

2 . The compound or the salt thereof according to claim 1 , wherein in the formula (I), a group represented by —O-L-R 12 is a group represented by the following formula (II):

where “n” represents an integer of from 0 to 10.

3 . The compound or the salt thereof according to claim 1 , wherein in the formula (I), a group represented by —O-L-R 12 is a hydroxyl group or a group selected from the following.

4 . The compound or the salt thereof according to claim 1 , wherein in the formula (I), a partial structure represented by

is a structure represented by the following formula:

where “m” represents an integer of from 1 to 3, and “q” represents 0 or 1.

5 . The compound or the salt thereof according to claim 1 , wherein R 10 and R 11 each represent an alkyl group, or are bonded to each other to form a cycloalkyl group.

6 . The compound or the salt thereof according to claim 1 , wherein in the formula (I), the amino acid B is Leu.

7 . The compound or the salt thereof according to claim 1 , wherein in the formula (I), the amino acid A is Met, Thr, His, Tyr, Phe, Ser, Gln, Arg, Lys, Nle, or MetO.

8 . The compound or the salt thereof according to claim 1 , wherein the compound is represented by the following general formula (III).

9 . A fluorescent probe, comprising the compound or the salt thereof of claim 1 .

10 . The fluorescent probe according to claim 9 , wherein the fluorescent probe is used for detecting calpain activity.

11 . A method of measuring calpain activity in a sample, comprising:
bringing the compound or the salt thereof of claim 1 and the sample into contact with each other; and measuring fluorescence from the compound of claim 1 after the contact.

12 . A diagnostic composition for a calpain activity-associated disease, comprising the compound or the salt thereof of claim 1 .

13 . The diagnostic composition according to claim 12 , wherein the calpain activity-associated disease is an eye disease, a muscle disease, diabetes, an inflammatory disease, an autoimmune disease, a neurological disease, a heart or blood vessel disease, a cancer, a brain tumor, an aging syndrome, progeria, an infectious disease, traumatic encephalopathy, Machado-Joseph disease, preeclampsia, or pulmonary fibrosis.

14 . The diagnostic composition according to claim 13 , wherein the eye disease is glaucoma, autosomal dominant neovascular inflammatory vitreoretinopathy, retinitis pigmentosa, age-related macular degeneration, retinal neuropathy or a retinal vascular occlusive disease associated with diabetes, retinal ischemia, or cataract.

15 . The diagnostic composition according to claim 14 , wherein the glaucoma is normal-tension glaucoma.

16 . The diagnostic composition according to claim 13 , wherein the cancer is a melanoma, breast cancer, colon cancer, kidney cancer, stomach cancer, cervical cancer, ovarian cancer, or soft tissue sarcoma.

17 . A method of providing information for diagnosis of a calpain activity-associated disease in a mammal subject, the method comprising:
bringing the compound or the salt thereof of claim 1 and a sample derived from the mammal subject into contact with each other; and measuring fluorescence from the compound or the salt thereof of claim 1 after the contact.

18 . A test method for determination of a therapeutic effect of a candidate compound on a calpain activity-associated disease, the method comprising:
preparing a sample from a calpain activity-associated disease model to which the candidate compound is not administered and a sample from the calpain activity-associated disease model to which the candidate compound is administered; bringing the compound or the salt thereof of claim 1 and each of the samples into contact with each other; and measuring fluorescence from the compound or the salt thereof of claim 1 after the contact.

19 . A method of providing information for monitoring of a calpain activity-associated disease in a mammal subject, the method comprising:
bringing the compound or the salt thereof of claim 1 and a sample derived from the mammal subject into contact with each other; and measuring fluorescence from the compound or the salt thereof of claim 1 after the contact.

20 . The fluorescent probe according to claim 9 , wherein the fluorescent probe is used in combination with a pharmaceutical composition for treating or preventing a calpain activity-associated disease, the pharmaceutical composition including a calpain inhibitor as an active ingredient, for determining a degree of an effect of the pharmaceutical composition in treatment or prevention of the disease with the pharmaceutical composition.

21 . The fluorescent probe according to claim 9 , wherein the fluorescent probe is used for examining in advance whether or not a treatment subject candidate or a prevention su

Assignees

• Tohoku Techno Arch Co Ltd
• Goryo Chemical Inc
• Senju Pharma Co

Inventors

• Nakazawa Toru
• Suzuki Yuki
• Oka Takayuki
• Uchida Takatoshi

Classifications

• C09B11/24

  Phthaleins containing amino groups {; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes} · CPC title

• C07K5/06052

  Val-amino acid · CPC title

• G01N33/6896

  Neurological disorders, e.g. Alzheimer's disease · CPC title

• C07D493/10

  Spiro-condensed systems · CPC title

• A61K49/0021

  the fluorescent group being a small organic molecule · CPC title