# US 2017/0285040 A1 · Ca ix - nir dyes and their uses

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- Number: US 2017/0285040 A1
- Document ID: US20170285040A1
- Status: Published application
- Kind: A1
- Application number: 15/461,361
- Filed: 2017-03-16
- Published: 2017-10-05
- Earliest claimed: 2016-03-16
- Assignees: On Target Laboratories, LLC
- Applicants: On Target Laboratories, LLC
- Inventors: Sumith A. Kularatne; Pravin Gagare; Ananda Kumar
- Web page: https://patentgrep.com/patent/US20170285040A1

## CPC

- A61B 5/0071: {by measuring fluorescence emission}
- A61B 1/000094: {extracting biological structures}
- A61B 1/043: {for fluorescence imaging}
- A61B 5/0073: {by tomography, i.e. reconstruction of 3D images from 2D projections (A61B5/0066 takes precedence)}
- A61B 5/0084: {for introduction into the body, e.g. by catheters (A61B5/1459 takes precedence)}
- A61B 5/7282: {Event detection, e.g. detecting unique waveforms indicative of a medical condition (cough events A61B5/0823; seizures A61B5/4094; sleep apnoea A61B5/4818)}
- A61P 35/00: Antineoplastic agents
- C07D 209/12: Radicals substituted by oxygen atoms
- C07D 311/90: with hydrocarbon radicals, substituted by amino radicals, directly attached in position 9
- C09B 23/0008: {substituted on the polymethine chain}
- C09B 23/0025: {the substituent being bound through an oxygen atom}
- C09B 23/0066: {the polymethine chain being part of a carbocyclic ring,(e.g. benzene, naphtalene, cyclohexene, cyclobutenene-quadratic acid)}
- C09B 23/166: {containing two or more nitrogen atoms (hydrazon dyes -CH=N-N- C09B26/02)}
- C12Y 402/01001: Carbonate dehydratase (4.2.1.1), i.e. carbonic anhydrase
- G01N 33/573: for enzymes or isoenzymes
- G01N 33/575: for cancer
- G01N 33/5758: {involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites}
- G01N 33/582: {with fluorescent label}
- G01N 33/583: {with non-fluorescent dye label}
- A61B 90/20: Surgical microscopes characterised by non-optical aspects
- A61B 90/30: Devices for illuminating a surgical field, the devices having an interrelation with other surgical devices or with a surgical procedure
- A61B 2090/373: {using light, e.g. by using optical scanners}
- A61B 2090/502: {Headgear, e.g. helmet, spectacles}
- A61B 2560/0431: Portable apparatus, e.g. comprising a handle or case
- A61K 49/0017: {Fluorescence in vivo}
- A61K 49/0032: {Methine dyes, e.g. cyanine dyes}
- G01N 2333/988: Lyases (4.), e.g. aldolases, heparinase, enolases, fumarase
- A61B: DIAGNOSIS; SURGERY; IDENTIFICATION (usefulness limited to only animals A61D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- C09B: ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES {, e.g. PIGMENTS}; MORDANTS; LAKES (fermentation or enzyme using processes to synthesise a desired chemical compound C12P)
- C12Y: ENZYMES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

The present disclosure relates to compounds that are useful as near-infrared fluorescence probes, wherein the compounds include i) a ligand that binds to the active site of carbonic anhydrase, ii) a dye molecule, and iii) a linker molecule that comprises an amino acid, amide, ureido, or polyethylene glycol derivative thereof. The disclosure further describes methods and compositions for making and using the compounds, methods incorporating the compounds, and kits incorporating the compounds.

## Claims

### Claim 1

A compound having the formula:
B-W-X-Y-Z
wherein B is a CA IX-targeted molecule;
W is an extended hydrophobic residue;
X is a hydrophobic spacer;
Y is an amino acid spacer; and
Z is a NIR dye.

### Claim 2 (depends on 1)

The compound of claim 1, wherein B is selected from the group consisting of a small molecule, ligand, inhibitor, agonist, and a derivative thereof.

### Claim 3 (depends on 1)

The compound of claim 1, wherein B is selected from the group consisting of
wherein n is 1, 2, 3 . . . , m is 0, 1, 2, 3, . . . , and p is 0, 1, 2,3, . . .
and derivatives thereof.

### Claim 4 (depends on 1)

The compound of claim 1, wherein B is 3-((2-(cyclooctylamino)-3,5,6-trifluoro-4-sulfamoylphenyl)sufonyl)proponoic acid or (C-SPA) or derivative thereof.

### Claim 5 (depends on 1)

The compound of claim 1, wherein W is a hydrophobic amino acid or moiety.

### Claim 6 (depends on 5)

The compound of claim 5, wherein W is a hydrophobic amino acid selected from the group consisting of Phe, Val, Leu, Ile, Trp, His, Arg, Lys, Asp, Glu, and derivative thereof.

### Claim 7 (depends on 1)

A compound of claim 1, wherein W is selected from the group consisting of:
neutral nonpolar amino acids including alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, methionine or aromatic group, cyclohexyl group, tyrosine, and derivatives thereof;
basic (positively charged) amino acids such as arginine, histidine, and lysine and derivative thereof; and
neutral polar amino acids, such as glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine and derivative thereof.

### Claim 8 (depends on 1)

A compound of claim 1 wherein W is an aromatic amino acid and derivative thereof.

### Claim 9 (depends on 1)

A compound of claim 1 wherein W is an hydrophobic residue and derivative thereof.

### Claim 10 (depends on 1)

A compound of claim 1 wherein W is charged.

### Claim 11 (depends on 1)

The compound of claim 1, wherein X is a hydrophobic linker.

### Claim 12 (depends on 1)

The compound of claim 1, wherein X is a length from 7 atoms to 14 atoms.

### Claim 13 (depends on 1)

The compound of claim 1, wherein X is selected from the group consisting of eight aminooctonoic acid (EAOA), polyethylene glycol (PEG), polyethylene amine (PEA), and N-amino-dPEG2 acid.

### Claim 14 (depends on 1)

A compound of claim 1, wherein X is selected from the group consisting of:
six aminohectanoic acid (SAHA), eight aminooctonoic acid (EAOA), polyethylene glycol (PEG), polyethylene amine (PEA) unit, a chain of 6 atoms, a spacer 6 atoms in length, a chain from 6 to 20 atoms in length, and a peptide comprising aryl or aryl alkyl groups, each of which is optionally substituted, and wherein one aryl or aryl alkyl group is about 6 to about 10, or about 6 to about 14 atoms, and the other aryl or aryl alkyl group is about 10 to about 14, or about 10 to about 15 atoms, or about 1 to about 20 atoms.

### Claim 15 (depends on 1)

The compound of claim 1 wherein X is variably charged.

### Claim 16 (depends on 1)

The compound of claim 1 wherein X is a peptide compromising positively charged amino acids (e.g. Arg, Lys, Orn) or quaternary amine containing amino acid.

### Claim 17 (depends on 1)

The compound of claim 1 wherein X has a negative charge.

### Claim 18 (depends on 1)

The compound of claim 1 wherein X has a positive charge.

### Claim 19 (depends on 1)

The compound of claim 1 wherein Y is selected from the group consisting of:
acidic amino acids selected from the group consisting of aspartic acid, glutamic acid and derivatives thereof;
basic amino acids selected from the group consisting of arginine, histidine, lysine, and derivatives thereof;
neutral polar amino acids selected from the group consisting of glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine and derivatives thereof; and
neutral nonpolar amino acids selected from the group consisting of alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, methionine, and derivatives thereof.

### Claim 20 (depends on 1)

The compound of claim 1 wherein Y is an aromatic amino acid or a derivative thereof.

### Claim 21 (depends on 1)

The compound of claim 1 wherein Y has a positive charge.

### Claim 22 (depends on 1)

The compound of claim 1 wherein Y has a negative charge.

### Claim 23 (depends on 1)

The compound of claim 1 wherein Y is an amino acid spacer with a chalcogen-containing side chain group.

### Claim 24 (depends on 1)

The compound of claim 1 wherein Y is an amino acid spacer with a sulfur-containing side chain group.

### Claim 25 (depends on 1)

The compound of claim 1 wherein Y is selected from the group consisting of cysteine, methionine, and an amino acid spacer with a sulfur-containing thiophenol moiety.

### Claim 26 (depends on 1)

The compound of claim 1 wherein Y comprises a tyrosine with a carbon isotope on the aromatic ring of tyrosine.

### Claim 27 (depends on 1)

The compound of claim 1 wherein Y comprises an amino acid with an aromatic ring with a hydrogen isotope.

### Claim 28 (depends on 1)

The compound of claim 1, wherein Z has a positive charge.

### Claim 29 (depends on 1)

The compound of claim 1, wherein Z has a negative charge.

### Claim 30 (depends on 1)

The compound of claim 1, wherein Z is selected from the group consisting of LS288, IR800, SP054, S0121, KODAK, S2076, S0456,
wherein R41, R42, R43, R44, R45, R46, R47, R48, ═H or SO3H; X═O, S, or N.

### Claim 31 (depends on 1)

The compound claim 1, wherein:
B comprises C-SPA or a derivative thereof;
X is selected from the group consisting of EAOA, PEG and PEA;
Y is selected from the group consisting of tyrosine, phenylalanine-tyrosine, histidine-tyrosine, phenylalanine-arginine-tyrosine, and histidine-tyrosine;
and Z comprises 50456.

### Claim 32

A compound having the structural formula:
or a pharmaceutically acceptable salt thereof, or isotopes thereof, wherein:
R19 represents a hydrogen or SO3H;
R20 represents a hydrogen, CH3, C3H6SO3−, C3H6SO3H or C4H8SO3−, or C4H8SO3H or C3H6N+(CH3)3;
R21 represents a carbon, optionally one or more sharing bonds,
R22 represents a carbon with optionally one or more sharing bonds;
R23 represents nitrogen, oxygen, or sulfur or no atom (direct C—C bond between aromatic ring and vinyl ring);
R24 is optional and when present represents aromatic substitution group to enhance the spectral properties such as increase brightness and stability of the vinyl ether bridge;
R25 is optional and when present
represents linkers with aromatic amino acids such as Phe, Trp, His or derivative thereof, cationic amino acids such Arg, Lys, or derivative thereof, anionic amino acids such as Asp, Glu or derivative of them, unnatural amino acids of aromatic/cationic/anionic acids or derivative thereof;
R26 is optional and when present represents a linear carbon chain, or polyethylene glycol linker, cationic linker, or derivative thereof;
R27 optional and when present represents hydrophobic moiety such as Phe, Val, Leu, Ile, Trp, His, Arg, Lys, Asp, Glu, or derivative thereof;
R28 represents hydrophobic linker; and
R29 is optional and when present represents aromatic substitution group to enhance the binding affinity, stability, hydrophobicity of the molecule such as F, NO2, or thereof, R30 optional and when present represents hydrophobic moiety such as Phe, Val, Leu, Ile, Trp, His, or derivative thereof or cyclic moiety such as cyclohexyl, cyclooctyl, or derivative thereof.

### Claim 33 (depends on 32)

The compound of claim 32 wherein said compound is selected from the group consisting of:

### Claim 34 (depends on 1)

The compound of claim 1 wherein the compound has an absorption and emission maxima between about 500 nm and about 900 nm.

### Claim 35 (depends on 1)

The compound of claim 1, wherein the compound is made to fluoresce after distribution thereof in the tissue cells.

### Claim 36 (depends on 1)

The compound of claim 1 wherein the compound is made to fluoresce by subjecting the compound to excitation light of near infrared wavelength.

### Claim 37 (depends on 1)

The compound of claim 1, wherein the compound has a binding affinity to carbonic anhydrase IX that is similar to the binding affinity of C-SPA.

### Claim 38 (depends on 1)

The compound of claim 1 wherein the compound is highly selective for targeting to a tumor cell.

### Claim 39 (depends on 1)

A compound of claim 1, wherein the compound comprises a linker composed of components X or W-X-Y.

### Claim 40 (depends on 39)

A compound of claim 39, wherein in the linker is releasable.

### Claim 41 (depends on 39)

A compound of claim 39, wherein the linker is at least about 6 atoms in length.

### Claim 42 (depends on 39)

A compound of claim 39, wherein the linker is between about 6 and about 20 atoms in length.

### Claim 43 (depends on 39)

A compound of claim 39, wherein the linker is at least about 10 angstroms (Å) in length.

### Claim 44 (depends on 1)

A method of optical imaging of a biological tissue that expresses CA IX, the method comprising:
(a) contacting the biological tissue with a composition of claim 1,
(b) allowing time for the compound in the composition to distribute within the biological target;
(c) illuminating the tissue with an excitation light of a wavelength absorbable by the compound; and
(d) detecting the optical signal emitted by the compound.

### Claim 45 (depends on 44)

The method of claim 44, wherein the signal emitted by the compound is used to construct an image.

### Claim 46 (depends on 44)

The method of claim 44, wherein the tissue is in a subject and the subject is an animal or human.

### Claim 47 (depends on 44)

The method of claim 44, wherein in step (a) two or more fluorescent compounds whose signal properties are distinguishable are contacted with the tissue, and optionally the tissue is in a subject.

### Claim 48 (depends on 43)

The method of claim 43, wherein the illuminating and detecting steps are performed using an endoscope, catheter, tomographic system, hand-held optical imaging system, surgical goggles, or intra-operative microscope.

### Claim 49 (depends on 1)

A method of identifying a target cell type in a biological sample comprising
a) contacting the biological sample with a compound of claim 1 for a time and under conditions that allow for binding of the compound to at least one cell of the target cell type; and
b) optically detecting the presence or absence of the compound in the biological sample,
wherein presence of the compound in detecting step b) indicates that the target cell type is present in the biological sample.

### Claim 50 (depends on 49)

The method of claim 49 wherein the target cell type is tissue to be imaged for diagnostic purposes.

### Claim 51 (depends on 49)

The method of claim 49 wherein the tissue is a tumor or a lymph node.

### Claim 52 (depends on 49)

The method of claim 49 wherein the target cell type is cancerous.

### Claim 53 (depends on 52)

The method of claim 52 wherein the cancer is selected from the group consisting of brain, breast, cervical, rectal and lung cancer.

### Claim 54 (depends on 1)

A method of performing image guided surgery on a subject comprising:
a) administering a composition comprising the compound of claim 1 under conditions and for a time sufficient for the compound to accumulate at a given surgical site;
b) illuminating the compound to visualize the compound using infrared light; and
c) performing surgical resection of the areas that fluoresce upon excitation by the infrared light.

### Claim 55 (depends on 1)

A method of diagnosing a disease in a subject comprising:
a) administering to a subject in need of diagnosis an amount of a compound of claim 1 for a time and under conditions that allow for binding of the compound to at least one cell of the target cell type;
b) measuring the signal from the compound of claim 1 present in the biological sample;
c) comparing the signal measured in b) with at least one control data set, wherein the at least one control data set comprises signals from the compound of claim 1 contacted with a biological sample that does not comprise the target cell type; and
d) providing a diagnosis of disease wherein the comparison in step c) indicates the presence of the disease.

### Claim 56 (depends on 1)

A kit comprising a compound of claim 1.