UA82684C2 - Hydrophilic, reacting in regard to thiol group cyanine dyes, the process for theirs preparation, conjugates of these dyes with biomolecules, the process for theirs preparation, and diagnostic set - Google Patents

Abstract

This invention relates to new fluorescence dyes from the class of cyanine dyes, especially to indotricarbocyanines with an absorption and fluorescence maximum in the spectral range of 700 to 900 nm, a thiol-specific reactive group, and three, preferably four, sulfonate groups, to an increase of water solubility as well as the production of dyes. This invention also relates to the conjugates of these dyes with biomolecules and uses thereof.

Description

The present invention relates to new fluorescent dyes from the class of cyanine dyes, in particular indotricarbocyanines with an absorption and fluorescence maximum in the spectral region from 700 to 90 Ohm, with a reaction group specific for the thiol group and three, preferably four, sulfonate groups, increasing solubility in water, as well as obtaining dyes. The present invention also relates to conjugates of these dyes with biomolecules and their use.

Recently, the use of light in medical diagnostics has gained importance (see, for example, Vioteaisai

RPpoiopish Naparook (Edyog t. Mo-Oipy)), SAS Rgevz5). A large number of various diagnostic methods for use in various medical fields, such as endoscopy, mammography, surgery and gynecology, have been developed during experimental research. Processes based on the application of light have high instrumental sensitivity and enable molecular detection and visualization of the smallest amounts of chromophores and/or fluorophores (MUeiz5iIedeg ei ai. (2001) MoIesshag Itadipo, Nadioiodu 219, 316-333).

Dyes that are injected into tissues as exogenous contrast media for the purpose of fluorescent diagnostics and visualization, especially fluorescent dyes with an absorption and fluorescence maximum in the spectral region of 700-900 nm (diagnostic tissue window), are of particular interest for use in ip mimo conditions. Protons of these wavelengths are relatively poorly absorbed by tissues and can therefore penetrate the tissues for several centimeters before the absorption process (mainly by the action of oxyhemoglobin and deoxyhemoglobin) stops the movement of light. Absorption can also occur with fluorescent dyes that are injected into the tissue, but which emit the absorbed energy as longer-wavelength fluorescent radiation.

Fluorescent radiation can be detected spectrally and enables the localization of dyes and correlation with the molecular structures to which the dye is attached (see in this connection Hisa, K. (2002) Sopigavi Adepiv tyug Oriisa! Itadipdo (Vemiem). In: Torisv ip Sitepi Spetivigu - Sopigavi Adepiv II (Ediyug: MU. Ktgaize), Moishte 222, zZriipdeg NeideIrego, pp. 1-31.|.

In order to achieve a diagnostically significant difference between diseased structures and healthy tissues, the injected dye should lead to a high, as possible, concentration difference between the tissues.

This can be done based on the physiological properties of the tumor (blood supply, localization kinetics, late removal). Conjugates consisting of fluorescent dyes with target-related biomolecules such as proteins, peptides, and antibodies can be used for molecular labeling of disease-specific structures. After injection, a certain part of these conjugates binds to such molecular target structures as receptors or matrix proteins, while the unbound part is excreted from the body. Thus, when performing fluorescent diagnostics, a higher concentration difference leads to greater visual contrast.

Fluorescent dyes of the class of cyanine dyes fall into the category of promising representatives and have been synthesized using many different structures. In particular, carbocyanines with indocarbocyanine, indodicarbocyanine, and indotricarbocyanine structures have high extinction coefficients and good fluorescence quantum yields (ispa, K. (2002) Sopigavi Adepiv og Oriisai!

Itadipd (Aemiemu). In: Torisv ip Sstepi Spetivigu - Sopigazi Adepiv II (Ediyug: MU. Kgaize), Moishte 222, Zriipdeg

Neideirego, pp. 1-31, and the references given in them).

GU application of UMO 96/17628| described the diagnostic process carried out under the conditions of ip mimo, using near-infrared radiation. In this case, water-soluble dyes and their biomolecular adducts with specific photo-physical and pharmacochemical properties are contrast media for fluorescent and trans-illumination diagnostics.

The synthesis of cyanine dyes with indocarbocyanine, indodicarbocyanine and indotricarbocyanine structures is well known in the art. Related relevant literature is presented, for example, in:

IViosopitsdaie Spet. 4, 105-111, 1993; Viosopitsdae Spet. 7, 356-62, 1996; Viosopipdage Spet. 8, 751-56, 1997; Suyuteyu 10, 11-19, 1989 and 11, 418-30, 1990; 9. Neyegosusi. Spent 33, 1871-6, 1996; 9. Ogd. Spent 60, 2391-5, 1995; Ouez apa Ridtepiv 17, 19-27, 1991, buez apa Ridtepiv 21, 227-34, 1993; 9. Ripogesis. Z, 153-155, 1993; and Apa!. Viospet. 217, 197-204, 1994).

Additional methods are described in patent publications. 5 4 981 977; 05 5 688 966; 05 5 808 044; MO 97/42976; MO 97/42978; MO 98/22146; UMO 98/26077; 1ER 0 800 8311.

In addition, indotricarbocyanines with other substituents have been synthesized and attached to biomolecules (described, for example, in Rpoiospet. Rpogobio!. 72, 234, 2000; Viosopiidaye Spet. 12, 44, 2001; Maishge Vioiespoi. 19, 237, 2001; y. Viotea . Oriiysz 6, 122, 2001; 9. Med. Spet. 45, 2003, 20021. Other examples can be found, in particular, in the publications of IMO 00/61194 ("5Zpop-Snaip Reriide YuOuye Sopitsdaisez az Sopigavi Adepiv Hog Oriiisa! Oiadpoviisv") , MO 00/71162, UMO 01/52746, UO 01/52743 and MO 01/621561.

However, the known indotricarbocyanines, which were previously used by specialists in the field of technology, have disadvantages that impair their effective use.

Low fluorescence quantum yields of indotricarbocyanines always occur after attachment to a biomolecule. Yes, Stireg et al. (Viosopipdaiye Spetp. 11, 696-704, 2000) note that for the commercially available Su7 dye, the loss of fluorescent efficiency occurs after attachment to the biomolecule.

WesKeg, etc. (Rpoiospet. Rpoiobioi. 72, 234, 2000) describe conjugates of indotricarbocyanine with NAZ5 and transferrin with fluorescence quantum yields of 2.995 or 2.895 and a deformed absorption spectrum in a physiological environment.

In addition, dye conjugates tend to aggregate. VeskKeg, etc. (Rpoiospet. Rpoiobio!. 72, 234, 2000) for conjugates of indotricarbocyanine with HA5 and transferrin and I sleep, etc. (Viosopiidage Spet. 12, 44-50, 2001) for receptor-binding peptides described them as having a deformed absorption spectrum in a physiological environment. These deformations indicate the formation of aggregations and the resulting fluorescence quenching. A similar problem exists in the case of inadequate water solubility of dyes.

As for reactive groups, there is also always the problem of inefficient access to derivatives. So,

Sipbeg, etc. (Viosopipdaiye Spetp. 11, 696-704, 2000) describe the use of Su7-bifunctional HA5-esters with two reactive groups, which can potentially lead to the formation of cross-links between two biomolecules. However, in the presence of two carboxyl groups in the molecule, synthetic access to derivatives with only one reactive group is difficult and leads to side products (for example, to the content of a portion of monoreactive NAb-esters Su7 of the unactivated and doubly activated Su?7 molecule).

Thus, there is a continuing need for efficient and easily obtainable cyanine dyes for fluorescent diagnostics, which have reduced or completely absent disadvantages mentioned above. In addition, these dyes should be sufficiently suitable for obtaining conjugates with biomolecules.

The first object according to the invention is an indotricarbocyanine dye of the general formula (I)

IN

Ya. re Her Trey shk v le: MN NI M v ! her and (her" in which

X means O, 5 or C, i.e. substituted in two places, while the substituents can be selected from methyl, ethyl, propyl, isopropyl and/or butyl; XU means CH»-CH»o or CH»-CHNo-CH»g; 7 means C-alkyl, while C atoms are optionally replaced by O or 5, or (о у

She

Vi-Va, independently of each other mean 5OzH or H, provided that at least three of Vi-Va mean

ЗОЗН, Б5 means -СО-МН-Нав-Но, -МН-С5-МН-На-Неа or -МН-СО-Нв8-Не, where Нв is selected from the group which contains unbranched C1-1 isalkyl in which carbon atoms optionally substituted with O or 5, and Not selected from

YAN ikh, Z and:

Her. - their 1 or У-о or acetyl chloride, acetide bromide, acetyl iodide, chloroacetamido group, iodoacetamido group, chloroalkyl, bromoalkyl, iodoalkyl, pyridyl disulfide and vinyl sulfoamide, and in which Не and Н7 stand for CH or are bound to radicals from alkyl to hexyl ring, which may optionally be substituted in the n-position by a Si-4 alkyl radical, and salts and solvates of this compound.

Preferred is the indotricarbocyanine dye according to the present invention, in which U means SNeo-CH»e; 7 means C-alkyl, while the carbon atoms are optionally replaced by O or 5, and in which Hn and A; mean CH, and salts and solvates of this compound.

Most preferred is the indotricarbocyanine dye of the present invention in which 7 is Si-alkyl.

Even more preferred is the indotricarbocyanine dye of the present invention, in which 9)

You and Av and A; linked to the hexyl ring through C3-alkyl.

Fluorescent dyes from the class of cyanine dyes, in particular indotricarbocyanines with an absorption and fluorescence maximum in the spectral region from 700 to 900 nm, a reactive group specific for a thiol group and three, preferably four, sulfonate groups, are thus objects of the present invention . The latter are used to increase solubility in water.

Surprisingly, it was recently found that indotricarbocyanines according to the invention with the above-mentioned structure (compact position of 3-4 sulfonate groups with sulfonatetyl radicals) have a high fluorescence quantum yield of 21595, and that the fluorescence quantum yield after attachment to biomolecules remains practically unchanged (the maximum loss is about 1090 ). In addition, the absorption spectra of the conjugates do not reveal any deformation in the near-infrared region of about 750 nm. Thus, dyes with good hydrophilicity, reduced aggregation and increased fluorescence quantum yield were obtained compared to standard indotricarbocyanines or Su7 derivatives with less than three sulfonate groups, particularly in the case of Su? and other known structures.

Another valuable aspect in the preparation of cyanine dyes for fluorescent diagnostics according to the invention relates to derivatives that have reactive functional groups that enable covalent binding to target-specific biomolecules. Suitable derivatives are, for example, MNO ethers and isothiocyanates (Viosopitsdage Spet. 4, 105-111, 1993; Viosopijdaie Spet. 8, 751-56, 1997|), which react with amino groups in such compounds as, for example, malonic acid imides, o-haloketones, or os-haloacetamides (Viosopindae Spet. 13, 387-391, 2002; Viosopiidae Spet. 11, 161-166, 2000), which react with thiol groups. Other bifunctional linkers (binding intermediate groups) can come from from the group consisting of arylene diisothiocyanates, alkylene diisothiocyanates, bis-m-hydroxysuccinimidyl ethers, hexamethylene diisocyanate and ether

M-(y-maleimidobutyryloxy)succinimide.

IU application of UMO 01/77229| described cyanine dyes with a combination of sulfoaryl groups, alkyl substituents in the meso-position of the methine chain, and at least one reactive group that enables attachment to biomolecules. However, embodiments relate to indodicarbocyanines (whose polymethine chain consists of 5 carbon atoms) and compounds that do not have reactive groups in the meso position.

GU application UMO 00/16810 ("Meag-Ipitagei RiIpogezzepsi Sopigazi Adepi apa Ripogezsepse Itadipo")| described indotricarbocyanines, for example, with substituents in the meso position of the C7-polymethine chain. It is not specified, however, how the reactive groups may be introduced or obtained.

Another aspect of the present invention relates to an indotricarbocyanine dye in which HA5 is COOH or MH."

The published derivatives are based mainly on SuZ3-, Sub5-, Su5,5- and Su7-based structures (commercially available from Ategepat RNagptasia Vioyesn; 05 5 268 486; SuZ - indocarbocyanine, Su5 - indodicarbocyanine, Su7 « - indotricarbocyanine). Derivatives with a reactive group relative to thiol are of particular interest because they enable direct binding to biotechnological cysteines, which occupy a specific position among biomolecules. The state of the art now concentrates mainly on

SuZz and Sub derivatives.

Especially preferred indotricarbocyanine dyes according to the invention are selected from dyes with formulas from (II) to (XX), which are presented below in Table 1.

Table 1: Preferred dyes according to the invention ele AND POM

Niryakovkh | she a : and, | Ho:

WHAT | Handicap in ! i WRITE she i i i i ' | yod same: yes ki y and vyh, V

Nervyad | Febui tia E

IF N and I

E RU | Ki, Y veshchyny Zk : : approx. a m t i det ! и H 3: essays by Я и и N

In | give your child N

Pirikanya | What is it ind di ik pt tu! і N I ih I EN I za zi is U also i

In p nna a i i CHI i

N | and oh all

E | That's Ma

Ilona | , eat! (Y ! i! Z i DYA hi shchi? 4 i i s Dn ЧЦЯ g Kdvktn, oVM i ! E shi ozh v s st i ' ! K, ra now rtfennnnennnneeedkeeeennnveneekkkkkkn young she

Mbhrnkhly p - telya i nyah N i sh

PO, J. oh and

Not them., they are still - hi stkokhtso shchi and gi

Between | . 2

N N : p.

No! and le 4 A and | HE I h

N and what and N them still ay My PTO E and Gayu them - Kt ! tires br N Sl. and N

Y Kot To i Kok : i : Eh N kan v TSI SCHI i : 3 s -x shchi ' : : 5 De: : ; ! What! и NYA Shchi : : et yi i : i i i x x uch i i | like Yod and Shk! I sittet tt teye I! Thursday And that | yati

HANDS 1 in med a SHU N: di pit ek prin di tt I

We are still chewing

Geunselyua with I : I : and N ! and: E. I think it's a lie; and m - in a nut:

I'm TOK, sleep: How:

Todith oh

In x N cad here is YAN ? ' : CODE et dm i M dini ; y I Z y y x - y : : : hee hee ! now about I word x;

KNU: r: | Z Z dan i tpzuskh I "x, still ke z-d Z i

Tunakhzya k: I know! sh t Y ! , 4 e r: Ї I,: Eh what's

With the help of VV

NksYa xx i eh h i y k -k ih : r : y yar y N y : y N

GIA G-y t U y Ko he xx : y I ho y t i sia j dekhto mine !

EN x Ka KE savan cha y v y yun H t eh ? and her and her: from Her and her I! t N: h ach y rohAy pike y Ko; še: WRITE; PISHI burned the lily;

He and nn ie!

Ted shSh

M nki p AKA zhzhA Kuk zhzhzhth yan flesh N

N m y y N ACTA AKA M AAAAAHAYEAAAAKA V X 5 !

Code:

PI! ши I yy, tyty I yatyanenia that K t Prakpl : S g | : What! | ! I

I N her - and

N ! she I

N

' g u i U Y ki

And I - - what ha

N ii obi xx ; one more time TE i i ev v i ke i i - i E also i i | Z Go in ді б зн х

And where are you? and | N /

N and No and | I :

N : :

N N and More ' | With lem! shi pope i: peck met ch t tt - y

I | donnnnetnntntttntotettnot - where are they.

BUT | and in

N sk net lyat he

N N tk, t i it t, I I.

Nryakia | r, and ; M. N

No! iv N.

Ii I:

And and and and! | r : N : : and eh more : and 5 you and Zhu ! Gozhoknnkh: tt i

N not a but a IN

And Ne yi TO be dum ikh ze : 1 x ve i

H 1 vn I : NO : E and N KE re and N y

In doses! and and BO ye

N N pour. - uknonnne shi 5 ho 1 . 4 and N c i

N N : And the Incas | And! another session! The thread

NU N: Z

N N and | : ! ; Sh

N yiv u t ZAY ooo SVO

N Tobi More K Kk

Go x I Sasha r '

N and Cheka : me : ! he and with skin Z! ! eat eat eat tk KZ ke Ya Ka I ! ! NK IIA ioobdyutt dk li y Z E se eh ni

I am 3 o TC I! 1 - ? !

N N | . i i sh-o i i N o i N . : i i : i de KAN i ! ab z gegeteniy and Y ona nivy avnik dra frtthntu nya! in. They have: ! pk and o

N I a ra sh kpc : ich sha in : oh eh" Because I she

V E N g N g : и KE I и и a И и у

With:

Io hi What Yi yan lov :

N Oh hi - dme yah i

ROOOYAH om: rin! obu TEZ ssh 3 The fief is still here. : x i E : i ; !

N N : i

Her I and : and N M and :

N Her I:

N Her: Fr

No more : : - 30

S nin -: that E i t m. SN; E kacha pi disc hi - ho i sdttitutku i i

Dead: p

N A o

And also;

De KT j shi Ya

N YN g "she:

N a: K i: le Pn a a i:

N I VU Ro bod stich children!

E M tiy s i NI y i V i ;

N HA xx m! . : pechhtttzhtttkttkihtnnnnnnni

Don't put your money in your money.

B and Z r. and My Ya

Mirvkhvh | breath still che er! i i N I GU pz : : Yi che !

And" In M ikh K: : ! In che I : ! and Ei ich i

In: i i i Mk i i H more

H xx : : where pc :

Ko t V dun di BO : : SV: Still Z ryh I! n: a and x. и и Я ' жо ИЗ ' t. и ох и : ИЗ !

ШЕ nie и в в : and В ОШ t і g what : ! And I r: and I

NE: her!

I also have:

H : o , : : :

N N | : i ven Yar :

And ::

N: no

N: p

N: p nd know how dlettyalyatnnyyatt tik palku; td Y: bevnuvh: PA AAU KA KAK Lu p i tyutny

The wise man is 5

TO US : and : to her with "

AND; sia and schi

INpnkozya o zi znan SE ' i N kh kh i E yi yi shen yi y | bu Z Zh i

N ! Article and

N : t I Her

H 3 x Y V

H 3 of them I : ! : ET V

Oh oh her ,

N Ryshe her x U ; | like her

Nish ische n z ische : : z 7 x ; Me IZ i : py ren : E - x : : Kei Zh Shchi s N shcha so : Z :

NOT in m v v a OR NI y

N y DN scha t isha:

H 1 Uh k | : i : ' : : ke i : : i i :

N : : : m i. :

I I st u: y pan tt

N K o i h i

TESHTTTTTTTT S

BEETLE and

In g th T i

In Ma oo av i

Gu emo: oh and children, Ya

Pernkma in ts Sha.

Z : m all : their ; in X.

E 3 E and : ше

Eat them and wait for KDM:

N n sko i karikh in V! That quieted down whose siy Z : : I MO tt se V

N t Kk i N x : : : ee i :

Where are we E

Cedar nnnnnnnnnnnnnnnnnnnnnaya on E

YHUNE i i s Ž i

There is a puppy: in: a puppy

NEbvnk pi shk

N : N Z Ch Z : pa And their and: : : A: and !

E KE. IS

And KU N

It's not like that

N iasch DE 5. Y o . more E and Tobi and and va: ! : Kleya M, : y - ! : :

RK i i je aa chik E N i nn o o p : : she i | y : : : shk : : : : S zo e E ' Y : N y

Our song! :

NOT ov: already zhk u u UK U U U U U puhu uh

Pryaklva: and vky be;

N x their - N . 3

I ray v s Sas Tr i

Elihad! их хи ин ик зи E and | Kok ik er (rik | H i y y N schu ! shi ШШшk o t t ko, drinking:

HM ts Uh sh- is

Another aspect of the present invention relates to a method of preparing the indotricarbocyanine dye of the present invention. In this case, a simple access via 4-substituted pyridines was found. Surprisingly, various 4-substituted pyridines can be converted in high yields using the Zincke reaction (reaction 2ipsKe-

Kopid, see Kotrr5 Spetiye And ehikop |Potrr5 Snetis! Oisiopagu|, 10 Eayop, p. 5067) in thiezo-substituted glutaconaldehyde-dianilide (precursor of cyanine dye).

In addition, the simple and effective synthesis of 4-substituted pyridines, the symmetric structures of the dyes according to this invention open up the possibility of certain derivatization with a reactive group selective for the thiol group in the symmetric meso position of the molecule.

In this way, further derivatization was carried out to compounds with a reactive group relative to thiol.

Functional reactive groups relative to thiol are, for example, "maleinimide (maleimide), chloroacetyl, bromoacetyl, iodoacetyl, chloroacetamide, bromoacetamide, iodoacetamide, chloroalkyl, bromoalkyl, iodoalkyl, pyridyl disulfide, and vinyl sulfonamide groups."

Another aspect of the present invention relates to the method of obtaining conjugates, which includes joining the indotricarbocyanine dye according to the present invention to a biomolecule. In the scope of this invention, the term "biomolecule" should be understood as any molecule of organic origin that has biological activity, in particular enzymatic activity, or binding substances of synthetic or biological origin, such as, for example, pharmaceutical agents, peptides, proteins, receptors or nucleic acids. In turn, the predominant biomolecules are proteins, such as enzymes, peptides, antibodies and antibody fragments (such as, for example, single chain, RAI, E(ar)", diarodiase, etc.), lipoproteins, nucleic acids such , such as oligonucleotides or polynucleotides from DNA or RNA, aptamers, RNA, and sugars such as, for example, mono-, di-, tri-, oligo- and polysaccharides.

The synthesis and biological characterization of conjugates of cyanine dyes with biomolecules such as peptides, antibodies and their fragments and proteins for fluorescent diagnosis of tumors under the conditions of IP mmo have been previously described in many publications. In this case, the above-mentioned Su3, Su5, Su5,5 and Su7 were mainly used. in connection with this, for example, Maishge Viogesppoi. 15, 1271, 1997; Sapseg Oeeyesi. Rhem. 22, 251, 1998; 9. Ittipo).

May 231, 239, 1999; Mashtge Viotesppoi. 17, 375, 1999; Mashge Medisipe 7, 743, 2001).

Another aspect of the present invention is related to the indotricarbocyanine dye conjugate obtained according to the method according to the invention with a biomolecule. This conjugate can be characterized by the fact that it includes a biomolecule as defined above, and as a biomolecule, at least one biomolecule selected from peptides, proteins, lipoproteins, antibodies or antibody fragments, nucleic acids such as, for example, oligonucleotides or polynucleotides DNA or RNA, aptamers, RNA, and sugars such as mono-, di-, tri-, oligo- and polysaccharides are most preferred. The attached protein can be characterized by being selected from the group of structural proteins or soluble proteins of the body. Absolutely preferred are serum proteins (for example, H5A), antibodies/antibody fragments, such as, for example, csEu-fragment or K(ab), as well as peptides, B5A, egg albumin or peroxidase derived from them.

Thus, from the state of the art, for example, antibodies are known that are directed against molecules that are intensively expressed in angiogenically active tissue and only at a very low level in the adjacent tissue | see UUO 96/016531.

Of particular interest are antibodies to receptors for vascular growth factors, receptors with endothelial cells, with which mediators of inflammation are bound, and matrix proteins, which are especially expressed during the formation of new vessels. Other antibodies and fragments of antibodies that oppose the matrix protein EOV-fibronectin and its conjugates according to the invention are preferred. EOV-fibronectin, also known as oncofetal fibronectin, is a splicing variant of fibronectin that is formed, in particular, around newly formed blood vessels in the process of angiogenesis. Antibodies 119, Ev, ARZ8 and others are particularly preferred

ARZ9 against EOV-fibronectin (Sapseg VNez 1999, 59, 347; ) Ittypo! Meiy 1999, 231, 239; Rgoivip Ehrg Rygii 2001, 21, 156).

The conjugate according to the invention is preferred, which is characterized by the fact that the indotricarbocyanine dye is attached to the biomolecule through a 5H group, in particular through a 5H group to cysteine. Not necessarily, even more preferably obtained from them are such antibodies and their fragments, which are obtained using recombinant technology so that at the C- or M-ends (within 1-10 extreme amino acids) they contain cysteine, which does not form any intramolecular 5-5-bridges, and therefore can be used to join dyes according to the invention.

Another aspect of the present invention relates to a diagnostic kit that includes an indotricarbocyanine dye of the present invention and/or a conjugate of the present invention. In addition, the kit may contain additional adjuvants for diagnosis, in particular of tumors, in the conditions of ip mimo. These adjuvants are, for example, suitable buffers, vessels, detection reagents or instructions for use. The kit preferably contains all materials for intravenous administration of dyes according to the invention.

The special content of such sets according to the invention is, for example, the following.

The first vessel contains an antibody (biomolecule) with a free ZH group and standard buffer/additives in the form of a solution or lyophilized material. Another vessel contains dyes according to the invention in the form of a solution (usual additives) or lyophilized material in a molar ratio of 0.1:1 (a 10-fold deficit in an equimolar amount). The contents of the vessel containing the dye are optionally mixed with buffer or distilled water, added to the contents of the vessel containing the biomolecule, incubated for 1-10 minutes, and used as an injection solution.

In another embodiment, the kit is a 2-chamber system (eg, a syringe) in which one chamber containing the antibody solution is physically separated by a collapsible partition from the second chamber containing the dye in solution or solid form. After breaking the partition, mixing and formation of an injection solution occurs.

The last aspect of the present invention relates to the use of the conjugate according to the invention as a fluorescent contrast medium for diagnosing tumors under IP mimo conditions. In this connection, the absorption maximum of Su7 lies in the region of 745 nm and is therefore particularly suitable for the detection of fluorescence in the conditions of ip mimo in the deeper layers of tissues (see above). However, Su? derivatives with reactive groups selective for the thiol group have not yet been described. In addition, the use of conjugates with antibodies to detect the boundaries of tumor spread has already been described in (MO 01/23005 (Apirodu YuOue Sopiidasez yug Vipaipd

Tagdei Bipisishgez oi Apdiodepeviv ip Ogaeeg yu Ipigaoregaimeiu Oerisi Tytog Reghirpegiez)), but without the use of promising dyes according to the invention.

The invention is further described in terms of the following examples and figures, which, however, do not limit it.

Figure 1 shows the standardized absorption and fluorescence spectra of the conjugate K11 (A) and K15 (B) (see Table 2) in PB5 (physiological solution with phosphate buffer).

Figure 2 shows the results of the visualized properties of the conjugates of example 24 according to the invention: substance: K15 conjugate, tumor: E9 teratocarcinoma in the lower right side of the mouse, dose: 5Onmol/kg body weight (data refer to the dye), excitation: 74Onm (diode laser), detection: SSO-camera (Natataivy) with 802.5--5 nm and a band-pass filter, time: before injection, 1 Th., bh. and 24 hours after the injection.

The position of the tumor is indicated by arrows.

Examples 1-16: Synthesis of indotricarbocyanine dyes with maleimide groups

Example 1: Trisodium 3,3-dimethyl-2-17-I(3,3-dimethyl-5-sulfonate-1-(2-sulfonatethyl)-ZH-indolium-2-yl|-4- (2-C2- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethylcarbamoyl|ethyl)hepta-2,4,b-trien-1-ylidene)-1-(2- sulfonateethyl)-2, 3-dihydro-1H-indole-5-sulfonate, internal salt (formula II) 6) and. MN r, ; ! re and ro, Makh - Yi o. x r A shi is 7 Ky i ve y x ;

what

OVO O0-58-0 Ma

FROM. o a) 1-(2-Sulfonateethyl)-2,3,3-trimethyl-ZN-indolenine-5-sulfonic acid, internal salt 10g (0.04ml) 2,3,3-trimethyl-ZN-indolenine-5-sulfonic acid (Viosopipade Spet 1993, 4, 105), 6.g (0.04ml) of 2-chloroethanesulfonic acid chloride and 4.2g (0.04ml) of triethylamine are mixed in 200ml of acetonitrile for 2 hours. The sediment is sucked off and dried. Yield 5.0g (3595 from theoretical). Apai Viospet. 1994217, 197. b) tert-butyl ether of 3-pyridin-4-ylpropionic acid 20 g (89 mmol) of tert-butyl-R,R-dimethylphosphonoacetate in 500 ml of THF is added dropwise at 0"C to a suspension of 3.9 g (98 mmol) of sodium hydride (6095-Na in mineral oil) in 250 ml of THF. After stirring for 1 hour at 0"C, a solution of 10 g (9 mmol) of pyridine-carbaldehyde in 5 ml of TIF is added dropwise, and the reaction mixture is stirred for 1 hour. at 0"7C and for 18 hours at room temperature. The solid substance that precipitated is removed by filtration, and the solution is concentrated by evaporation. The residue is dissolved in isopropanol when heated, the insoluble part is filtered off, and the solution is cooled to 0"C for crystallization. The solid substance is filtered, mixed with hexane, filtered off the hexane and dried. The intermediate product (15.3 g) was hydrogenated in 150 ml of ethanol with 0.15 g of palladium 10956 on activated carbon for 2 h. The catalyst is filtered off, the solution is concentrated by evaporation, and the residue is filtered on silica gel (the mobile phase is diethyl ether). 13.0 g of slightly yellow oil are obtained (yield 71905 from theoretical). c) 3-(2-(tert.-Butyloxycarbonyl)ethyl|glutaconaldehyde-dianilide-hydrobromide

A solution of 10 g (48 mmol) of tert-butyl ether of 3-pyridin-4-ylpropionic acid in 150 ml of diethyl ether is mixed with 8.9 g (9 mmol) of aniline and then mixed at 0"C with a solution of 5.4 g (48 mmol) of cyanobromine in 2 ml of diethyl ether. After stirring at 0"C, the red solid formed is filtered off, washed with ether and dried in a vacuum.

Yield: 20.3g (9295 from theoretical). d) Three sodium 3,3-dimethyl-2-17-3,3-dimethyl-5-sulfonate-1-(2-sulfonatethyl)-ZH-indolium-2-yl1|-4-(2-carboxyethyl)hepta- 2,4,b-trien-1-ylidene)-1-(2-sulfonatethyl)-2,3-dihydro-1H-indole-5-sulfonate, inner salt

A suspension of 1.0 g (2.2 mmol) of 3-(2-(tert-butyloxycarbonyl)ethyl|-glutaconaldehyde-dianilide hydrobromide (example) and 1.5 g (4.4 mmol) of 1-(2-sulfonateethyl)-2,3 ,3-trimethyl-ZN-indolenine-5-sulfonic acid (example 1a) in 20 ml of acetic anhydride and 5 ml of acetic acid are mixed with 0.75 g (9.1 mmol) of sodium acetate and stirred for an hour at 120"C. After cooling mixed with diethyl ether, the precipitated solid was filtered, purified by chromatography (AP-C18-silica gel, mobile phase water/methanol) and lyophilized (0.5 g). Deprotection is carried out by mixing the intermediate product in 4 ml of dichloromethane/1 ml of trifluoroacetic acid for 1 hour. After concentration by evaporation and chromatographic purification (AP-C18-silica gel, mobile phase water/methanol), 0.45 g (2395 from the theoretical) of a blue lyophilizate is obtained. d) Trisodium 3,3-dimethyl- 2-17-13,3-dimethyl-5-sulfonate-1-(2-sulfonatethyl)-3H-indolium-2-yl|-4-(2-C2-(2,5-dioxo-2,5-dihydro -1H-pyrrole- 1-yl)ethylcarbamoyl)ethyl)hepta-2,4,6-trien-1-ylidene)-1-(2-sulfonatethyl)-2,3-dihydro-1H-indole-5-sulfonate, inner salt

0.4 g (0.45 mmol) of the compound named in the title of example d, and 45 mg (0.45 mmol) of triethylamine are dissolved in 10 ml of DMF, mixed at 0"C with 0.15 g (045 mmol) of TVTI and stirred for 10 minutes. Then the solution is added 0.17 g (O0.68 mmol) of M-(2-aminoethyl)maleimide-trifluoroacetate (Ipi. 9U. Reri. Rgoieip Nev. 1992, 40, 445) and 8 mg (0 bdmmol) of triethylamine in 0.5 ml of DMF, and stirred for h at room temperature. Then 10 ml of diethyl ether is added, the solid is separated by centrifugation, dried and purified by chromatography (AP C-18 silica gel, methanol/water gradient). Yield: 0.30 g of blue lyophilizate (6595 from theoretical).

Elemental analysis: Calculated: C 47.24; H 4.26; M 5.51; 512.61; Ma 6.78

Found: C 47.74; H 4.47; M 5.40; 511.99; Ma 7.02

Example 2: Trisodium 3,3-dimethyl-2-(7-I(3,3-dimethyl-5-sulfonate-1-(2-sulfonatethyl)-ZH-indolium-2-yl|-4- (2-116 -(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexylcarbamoyl)ethyl)hepta-2,4,6-trien-1-ylidene)-1-(2- sulfonateethyl)-2 ,3-dihydro-1H-indole-5-sulfonate, internal salt (formula IP)

SN: tires tn kt 3 are in. Ve and oo

UST LIKE au ey y y ut y poh otvko en che in Z

The synthesis is carried out similarly to example Td with 0.4g (0.45mmol) of the compound named in the title of example 1g and 0.21g (0.6d8mmol) of M-(6b-aminohexyl)malein midtrifluoroacetate (Ipi. U. Reri. Rgoiveip Nez. 1992, 40, 445). Yield: 0.38g of blue lyophilizate (8195 from theoretical).

Elemental analysis: Calculated: C 49.25; H 4.79; M 5.22; 5 11.95; Ma 6.43 Found: C 48.96; H 4.92; M 5.32;

From 11.88; Ma 6.56

Example 3: Trisodium 3,3-dimethyl-2-17-I(3,3-dimethyl-5-sulfonate-1-(2-sulfonatethyl)-3H-indolium-2-yl|-4- (2-ShCh13- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4,7,10-trioxatridecylcarbamoyl)ethyl)hepta-2,4,b-trien-1-ylidene)-1- (2-sulfonatethyl)-2,3-dihydro-1H-indole-5-sulfonate, inner salt (formula IM)

o n h

Oh oh;

G Y in (9) zl K- Hre dk 8 Sorym KK and Ma o-i-v sni nk o 19)

The synthesis is carried out similarly to the poison example with 0.4 g (0.45 mmol) of the compound named in the title of example 1 g and 0.28 g (0.68 mmol) of M-(13-amino-4,7,10-trioxatridecyl)maleimide trifluoroacetate (IPi . in). Rary. Rheumatology Nez., 1992, 40, 445). Yield: 0.27 g of blue lyophilizate (5195 from theoretical).

Elemental analysis: Calculated: C 49.97; H 5.05; M 4.76; 5 10.89; Ma 5.86

Found: C 49.22; H 5.16; M 4.62; 510.67; Ma 5.66

Example 4: Trisodium 3,3-dimethyl-2-17-3,3-dimethyl-5-sulfonate-1-(2-sulfonatethyl)-3H-indolium-2-yl|- 4-(4-C2-(2 ,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)y-ethylcarbamoylbutyl)hepta-2,4b-trien-1-ylidene)-1-(2- sulfonateethyl)-2,3-dihydro -S-indole-5-sulfonate, internal salt (formula M) about ! I a av t u ra y c) c) G i ots y PO zhen m Shk - tov o-Vko o-5-o ce o Go a) (3-tert.-Butoxycarbonylpropyl)-triphenylphosphonium bromide 50g (0.30ml ) of 4-bromobutyric acid is mixed dropwise in 400 ml of THF at -40"C with 187 g (0.89 ml) of trifluoroacetic anhydride. After 30 minutes of stirring at -40 "C, 400 ml of a mixture of tert-butanol/30 ml of THF is added dropwise for an hour . After 1 week stirring at room temperature, the reaction mixture is transferred to an ice-cooled sodium carbonate solution, the aqueous phase is extracted three times with diethyl ether, the organic phase is dried over sodium sulfate and concentrated by evaporation. The residue is distilled in a vacuum (boiling point 727°C/0.9mbar; yield: 41g). To form a phosphonium salt, the reaction is carried out by heating under reflux 41g (0.13ml) of the intermediate product, 44.bg (0.17ml) of triphenylphosphine and 32.5 g (0.3 bml) of sodium bicarbonate in 250 ml of acetonitrile for 20 h. The reaction mixture is filtered, concentrated by evaporation, and the residue for crystallization is mixed with diethyl ether.

Yield: 58.5 g (4095 of theoretical, relative to 4-bromobutyric acid) of a white solid. b) tert-Butyl ether of 5-pyridin-4-ylpentanoic acid

A solution of 14g (28mmol) (3-tert-butoxycarbonylpropyltriphenylphosphonium bromide (example 4a) in 100ml of anhydrous THF is mixed at -40"C in a vacuum for 20min with 17.5ml (28mmol) of butyllithium (1.6-molar in hexane) and stirred for 1 h at -40"C. A solution of 2.78 g (2.6 mmol) of 4-pyridinecarbaldehyde in 20 ml of THF is added dropwise and stirred for 16 h at room temperature, then transferred to ice water, the aqueous phase is extracted three times with diethyl ether, the organic phases are dried over sodium sulfate and concentrated by evaporation. After chromatographic purification (silica gel, mobile phase hexane/ethyl acetate)), the product is obtained as an E,2-mixture (4:11 according to NMR data; 5.0 g). For hydrogenation of the double bond, the intermediate product is dissolved in 200 ml of methanol and mixed with 100 mg of Ryu" catalyst at room temperature in a hydrogen atmosphere. After filtration and concentration by evaporation, a yellow oil is obtained. Yield: 4.9g (74905 from theoretical). c) 3-(4-tert-Butyloxycarbonyl)butyl|glutaconaldehyde-dianilide hydrobromide

A solution of 4.0 g (17 mmol) of tert-butyl ether of 5-pyridin-4-ylpentanoic acid in 35 ml of diethyl ether is mixed with 3.2 g (3 mmol) of aniline and then at 0"C with a solution of 1.9 g (17 mmol) of cyanogen bromide in 3 ml of diethyl ether. ether

After mixing during the Agreement. at 0"C, the red solid that was formed is filtered off, washed with ether and dried in a vacuum. Yield: 7.9 g (9595 from theoretical). d) Three sodium 3,3-dimethyl-2-17-I3,3-dimethyl -5-sulfonate-1-(2-sulfonatethyl)-ZN-indolium-2-yl1|-4-(4-carboxybutyl)hepta-2,4,6-trien-1-ylidene)-1-(2-sulfonatethyl) )-2,3-dihydro-1H-indole-5-sulfonate, inner salt

The synthesis is carried out similarly to example 1g from the compound named in the title of example 4c (2.5 mmol) and 1-(2-sulfonatethyl)-2,3,3-trimethyl-ZN-indolenine-5-sulfonic acid (bmmol). Yield: 0.85 g (3795 from theoretical) of blue lyophilisate. e) Three sodium 3,3-dimethyl-2-(7-3,3-dimethyl-5-sulfonate-1-(2-sulfonatethyl)-3H-indolium-2-yl1-4-(4-(2- 2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-ethylcarbamoylbutyl)hepta-2,4,6-trien-1-ylidene)-1-(2-sulfonateethyl)-2,3- dihydro-1H-indole-5-sulfonate, internal salt

The synthesis is carried out similarly to the example of food from 0.4 g (0.4 mol) of the compound named in the title of example 4 g.

Yield: 0.31 g (69905 from theoretical) of blue lyophilisate.

Elemental analysis: Calculated: C 48.27; H 4.53; M 5.36; 5 12.27; Ma 6.60

Found: C 48.01; H 4.44; M 5.56; 512.10; Ma 6.81

Example 5: Trisodium 3,3-dimethyl-2-(17-13,3-dimethyl-5-sulfonate-1-(2-sulfonatethyl)-ZH-indolium-2-yl|-4-(4-16-( 2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-hexyl-carbamoylbutyl)hepta-2,4,b-trien-1-ylidene)-1-(2-sulfonatethyl)-2,3- dihydro-1H-indole-5-sulfonate, internal salt (formula MI)

O- - lived) vva zrgu o a a 9 ne dk an i nya shchi me oto bsh8-o I and Ge Ma

The synthesis is carried out similarly to the example of poison from 0.4 g (0.4 mmol) of the compound named in the title of example 1g and 0.20 g (0.6 mmol) of M-(b-aminohexyl)maleimide of trifluoroacetic acid. Yield: 0.35 g of blue lyophilizate (7495 from theoretical).

Elemental analysis: Calculated: C 50.17; H 5.03; M 5.36; 5 11.65; Ma 6.26

Found: C 49.83; H 4.89; M 5.34; 5 12.05; Ma 6.42

Example 6: Trisodium 3,3-dimethyl-2-17-I(3,3-dimethyl-5-sulfonate-1-(2-sulfonatethyl)-ZH-indolium-2-yl|-4- (4-413- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4,7,10-trioxatridecylcarbamoyl)butyl)hepta-2,4,b-trien-1-ylidene)-1- (2-sulfonatethyl)-2,3-dihydro-1H-indole-5-sulfonate, inner salt (formula MII) (9) i t

Or get out or go

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HOW AYK o Y i o. ad aoi shchi c)

She t Ma, a-8ho bhv-o 00 y y ma

The synthesis is carried out similarly to example Id from 0.4 g (0.4 mmol) of the compound named in the title of example g and 0.30 g (0.72 mmol) of M-(13-amino-4,7,10-trioxatridecyl)maleimide-trifluoroacetate . Yield: 0.27 g of blue lyophilizate (5295 from theoretical).

Elemental analysis: Calculated: C 49.83; H 5.27; M 4.65; 5 10.64; Ma 5.72

Found: C 49.45; H 5.19; M 4.66; 5 10.85; Ma 5.80

Example 7: Trisodium 3,3-dimethyl-2-(17-3,3-dimethyl-5-sulfonate-1-(2-sulfonatethyl)-3H-indolium-2-yl|-4-(6-Х2-( 2,5-dioxo-2,5-dihydro-1H-trol-1-yl)-ethylcarbamoylhexyl)hepta-2,4,b-trien-1-ylidene)-1-(2-sulfonatethyl)-2,3- dihydro-1H-indole-5-sulfonate, inner salt (formula MI) o)

Oh guys

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Oh in H reshe zo o» s zhk | p, dk | (3-tert.-Butoxycarbonylentylutriphenylphosphonium bromide) Preparation is carried out similarly to that described in example 4a, while the intermediate product is tert-butyl ether Z-pyridine-4 -ylpropionic acid is introduced into the reaction in the form of a crude product. 79g of the product (6995 from theoretical) are obtained in the form of a viscous colorless oil from 50g of b-bromohexanoic acid. b) tert-Butyl ether of 7-pyridin-4-ylheptanoic acid

Preparation is carried out similarly to that described in example 46. tert-Butyl ether of 7-pyridin-4-ylheptanoic acid (7.5 g, 6595 from theoretical) is obtained as a yellow oil from 25 g (48.7 mmol) (3-tert.-butoxycarbonylpentyl) -triphenylphosphonium bromide (example 7a). c) 3-(4-tert-Butyloxycarbonyl)hexyl|glutaconaldehyde-dianilide hydrobromide

A solution of 5.0 g (19 mmol) of tert-butyl ether of 5-pyridin-4-ylheptanoic acid in 30 ml of diethyl ether is mixed with 3.6 g (3 mmol) of aniline and then at 0"C with a solution of 2.1 g (19 mmol) of cyanide bromide in 5 ml diethyl ether.

After stirring for 2.5 hours. at 0"C, the red solid that formed is filtered off, washed with ether and dried in a vacuum. Yield: 8.9 g (9195 from theoretical). d) Three sodium 3,3-dimethyl-2-17-I3,3-dimethyl -5-sulfonate-1-(2-sulfonatethyl)-ZN-indolium-2-yl1|-4-(6-carboxyhexyl)hepta-2,4,b-trien-1-ylidene)-1-(2-sulfonatethyl) )-2,3-dihydro-1H-indole-5-sulfonate, inner salt

The synthesis is carried out similarly to example 1g from the compound named in the title of example 4c (3mmol) and 1-(2-sulfonatethyl)-2,3,3-trimethyl-3H-indolenine-5-sulfonic acid (bmmol). Yield: 1.5 (54905 from theoretical) blue lyophilizate. e) Trisodium 3,3-dimethyl-2-17-13,3-dimethyl-5-sulfonate-1-(2-sulfonatethyl)-ZH-indolium-2-yl|-4-(6-C2- (2, 5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-ethylcarbamoyl)hexyl)hepta-2,4,b-trien-1-ylidene)-1-(2-sulfonatethyl)-2,3- dihydro-1H-indole-5-sulfonate, internal salt

The synthesis is carried out similarly to example Id with 0.4 g (0.43 mmol) of 4 g of the compound named in the title of the example.

Yield: 0.31 g (6995 from theoretical) blue lyophilisate.

Elemental analysis: Calculated: C 49.25; H 4.79; M 5.22; 5 11.95; Ma 6.43

Found: C 48.98; H 4.86; M 5.12; 5 11.76; Ma 6.77

Example 8: Trisodium 3,3-dimethyl-2-17-I(3,3-dimethyl-5-sulfonate-1-(2-sulfonatethyl)-ZH-indolium-2-yl|-4- (6-(6 -(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-hexylcarbamoyl)hexyl)hepta-2,4,6-trien-1-ylidene)-1-(2-sulfonatethyl)- 2,3-dihydro-1H-indole-5-sulfonate, inner salt (formula IX) and M yt

T / y g

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The synthesis is carried out similarly to example Id from 0.5 g (0.53 mmol) of the compound named in the title of example 7g and 0.23 g (0.75 mmol) of M-(6b-aminohexyl)maleimide-trifluoroacetate. Yield: 0.42 g of blue lyophilizate (7095 from theoretical).

Elemental analysis: Calculated: C 51.05; H 5.27; M 4.96; 5 11.36; Ma 6,11

Found: C 50.74; H 5.55; M 4.76; 511.38; Ma 6.35

Example 9: Trisodium 3,3-dimethyl-2-17-I(3,3-dimethyl-5-sulfonate-1-(2-sulfonatethyl)-ZH-indolium-2-yl|-4- (6-113- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4,7,10-trioxatridecylcarbamoyl)hexyl)hepta-2,4,b-trien-1-ylidene)-1- (2-sulfonatethyl)-2,3-dihydro-1H-indole-5-sulfonate, inner salt (formula X) and -

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The synthesis is carried out similarly to the example of poison from 0.5 g (0.53 mmol) of the compound named in the title of example g and 0.44 g (1.0 b mmol) of M-(13-amino-4,7,10-trioxatridecyl)maleinmid-trifluoroacetate . Yield: 0.24 g of blue lyophilizate (3795 from theoretical).

Elemental analysis: Calculated: C 50.64; H 5.48; M 4.54; 5 10.40; Ma 5.59

Found: C 50.30; H 5.56; M 4.34; 510.15; Ma 5.73

Example 10: Trisodium 3,3-dimethyl-2-(7-13,3-dimethyl-5-sulfonate-1-(2-sulfonatethyl)-ZH-indolium-2-yl1-4-(5-C2-(2 ,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-ethylcarbamoyl)-3-oxapentyl)hepta-2,4,6-trien-1-ylidene)-1-(2-sulfonatethyl) -2,3-dihydro-1H-indole-5-sulfonate, inner salt (formula XI) (o) o;

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C Still 0-80 o-8-0o M 8 0 Ma a) tert.--Butyl ether of 3-oxa-6-(4-pyridinyl)hexanoic acid Solution 75g (0.4ml) 3-(4-pyridinyl)- 1-

of propanol in 400 ml of toluene/50 ml of THF is mixed with 10 g of tetrabutylammonium sulfate and 350 ml of 3295% sodium hydroxide solution. Then 123 g (0.68 ml) of tert-butyl ether of bromoacetic acid is added dropwise and stirred for 18 hours. at room temperature. The organic phase is separated, the aqueous phase is extracted three times with diethyl ether. The combined organic phases are washed with a solution of MaSi, dried over sodium sulfate and concentrated by evaporation. After chromatographic purification (silica gel, mobile phase: hexane: ethyl acetate solution), 5 g of product are obtained as a brownish oil (4195 from theoretical). b) 3-(4-Oxa-5-(tert-Butyloxycarbonyl)pentyl|glutaconaldehyde-dianilide hydrobromide

A solution of 5.0 g (2 mmol) tert-butyl ether of 3-oxa-6-(4-pyridinyl)-hexanoic acid in bOml of diethyl ether is mixed with 3.7 g (40 mmol) of aniline and then at 0"C with a solution of 2.2 g (20 mmol) of cyanide bromide in ml of diethyl ether. After stirring for 17 hours at 0"C, the solid substance formed is filtered off, washed with ether and dried in a vacuum. Yield: 8.5 g (8595 from theoretical) of solid purple color. c) Trisodium 3,3-dimethyl-2-(7-3,3-dimethyl-5-sulfonate-1-(2-sulfonatethyl)-ZH-indolium-2-yl|-4-(6-carboxy-4- oxhexyl)hepta-2,4,b-trien-1-ylidene)-1-(2-sulfonatethyl)-2,3-dihydro-1H-indole-5-sulfonate, inner salt

A suspension of 3.0 g (bmmol) 3-(2-(tert.-butyloxycarbonyl)ethyl|-glutaconaldehyde-dianilide hydrobromide (Example 106) and 4.2 g (12 mmol) 1-(2-sulfonateethyl)-2,3,3- of trimethyl-ZN-indolenine-5o--sulfonic acid (example TA) in 5O0ml of acetic anhydride and 1Oml of acetic acid are mixed with 2.5g (ZOmmol) of sodium acetate and stirred for 50 minutes at 120"C. After cooling, they are mixed with diethyl ether, the precipitated solid was filtered off, precipitated by absorption in acetone and dried under high vacuum. After chromatographic purification (AP-C18-silica gel, mobile phase water/methanol), the methanol was removed in vacuo and lyophilized (0.5 g). After concentration evaporation and chromatographic purification (AP-C18-silica gel, mobile phase water/methanol), the title compound is immediately obtained.

Yield: 2.3 g (4195 from theoretical) of blue lyophilisate. d) Trisodium 3,3-dimethyl-2-17-13,3-dimethyl-5-sulfonate-1-(2-sulfonatethyl)-ZH-indolium-2-yl|-4-(5-C2- (2, 5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-ethylcarbamoyl)-3-oxapentyl)hepta-2,4,b-trien-1-ylidene|-1-(2-sulfonatethyl)-2 ,3-dihydro-1H-indole-5-sulfonate, inner salt

The synthesis is carried out in the same way as in Example 10 from 1.0 g (1.1 mmol) of the compound named in the title of Example 10c.

Yield: 0.85 g of blue lyophilizate (7395 from theoretical).

Elemental analysis: Calculated: C 47.54; H 4.46; M 5.28; 5 12.09; Ma 6.50

Found: C 47, 97; H 4.65; M 5.10; 5 12.02; Ma 6.68

Example 11: Trisodium 3,3-dimethyl-2-(7-13,3-dimethyl-5-sulfonate-1-(2-sulfonatethyl)-ZH-indolium-2-yl1-4-(5-116-( 2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl-hexyl|carbamoyl)-3-oxapentyl)hepta-2,4,6-trien-1-ylidene)-1-(2-sulfonatethyl )-2,3-dihydro-1H-indole-5-sulfonate, inner salt (formula XII)

Z o Z

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The synthesis is carried out similarly to the example of poison from 0.5 g (0.55 mmol) of the compound named in the title of example 10, and 0.23 g (0.75 mmol) of M-(b-aminohexyl)maleimide-trifluoroacetate. Yield: 0.42 g of blue lyophilisate (6890 from theoretical).

Elemental analysis: Calculated: C 49.46; H 4.96; M 5.01; 5 11.48; Ma 6.17

Found: C 48.95; H 5.21; M 5.22; 511.23; Ma 6.60

Example 12: Trisodium 3,3-dimethyl-2-(7-3,3-dimethyl-5-sulfonate-1-(2-sulfonateethyl)-3H-indolium-2-yl)-4-(5-C13-( 2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4,7,10-trioxatridecylcarbamoyl)-4-oxapentyl)hepta-2,4,6-trien-1-ylidene)- 1-(2-sulfonatethyl)-2,3-dihydro-1H-indole-5-sulfonate, internal salt (formula XIII) (c) certain

The synthesis is carried out similarly to the example of poison from 0.5 g (0.55 mmol) of the compound named in the title of example 10c and 0.Abg (1.06 mmol) of M-(13-amino-4,7,10-trioxatridecyl)maleimide-trifluoroacetate . Yield: 0.34 g of blue lyophilizate (5695 from theoretical).

Elemental analysis: Calculated: C 49.17; H 5.20; M 4.59; 5 10.50; Ma 5.65

Found: C 49.34; H 5.32; M 4.45; 510.28; Ma 5.56

Example 13: Trisodium 3,3-dimethyl-2-(2-(1-|3,3-dimethyl-5-sulfonate-1-(2-sulfonatethyl)-ZH-indolium-2-

yl1-vinylene)-2-I4-(2-C2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethylcarbamoyl)ethyl)-phenoxy|cyclohex-1-en-3- ylidene)ethylidene|-1-(2-sulfonatethyl)-2,3-dihydro-1H-indole-5-sulfonate, internal salt (KHIM formula) n man ) / o i (c) o o o o

Sh m" and tea - b o-8-0 o-8-07 ma and M ma a) Three sodium 3,3-dimethyl-2-(2-(141I3,3-dimethyl-5-sulfonate-1-( 2-sulfonateethyl)-ZH-indolium-2-yl|vinylene)-2h-chlorocyclohex-1-en-3-ylidene)ethylidene|-1-(2-sulfonateethyl)-2,3-dihydro-1H-indole-5 -sulfonate, inner salt 5.0 g (14.4 mmol) 1-(2-sulfonatoethyl)-2,3,3-trimethyl-ZN-indolenine-5-sulfonic acid (example Ta) and 2.6 g (7.2 mmol) M -(3-(anilinomethylene)-2-chloro-1-cyclohexen-1-ylylmethylene) aniline hydrochloride (Alihydrin Sotrapu) is heated under reflux together with 2.5 g (30 mmol) of anhydrous sodium acetate in 100 ml of methanol for 1 hour, cooled, mixed with 150 ml of diethyl ether and stirred overnight. The precipitate was suctioned off, dried and purified by chromatography (silica gel, gradient: dichloromethane/methanol). Yield: 3.8 g of a blue solid (5895 from theory). b) Trisodium 3,3- dimethyl-2-(2-(1-|3,3-dimethyl-5-sulfonato-1-(2-sulfonatoethyl)-ZH-indolium-2-yl|-vinylene)-2-(4-(2-carboxyethyluphenoxy) |cyclohex-1-en-3-ylidene)ethyl den|-1-(2-sulfonatoethyl)2,3-dihydro-1H-indole-5-sulfonate, inner salt 0.37g (2.2mmol) of 3-(4-Hydroxyphenyl)propionic acid in 30ml DMF is mixed with 0 ,18 g (4.5 mmol) of sodium hydride (6095 mineral dispersion). After stirring at room temperature for 30 hours, the mixture is cooled to 0"C, a solution of 2.0 g (2.2 mmol) of the compound named in the title of Example 12a in 100 ml of DMF is added dropwise and stirred for 2 hours at room temperature. The mixture is quenched with dry ice and the solvent is removed in vacuum. The residue is dissolved in methanol, mixed with 200 ml of ether, and the solid precipitate is filtered off. Chromatographic purification is carried out (silica gel, gradient: ethyl acetate/methanol). Yield: 1.9 g of a blue solid (8395 from theoretical). c) Trisodium 3.3 -dimethyl-2-|2-(1-I3,3-dimethyl-5-sulfonate-1-(2-sulfonatethyl)-ZH-indolium-2-yl1-vinylene)-2-(4-(2-C2- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethylcarbamoyl)ethyl)-phenoxy|cyclohex-1-en-3-ylidene)ethylidene1-1-(2-sulfonatethyl)-2 ,3-dihydro-1H-indole-5-sulfonate, inner salt

0.1 mg (0.10 mmol) of the compound named in the title of example 126 is reacted with TVTI and M-(2-aminoethyl)maleimide--rifluoroacetate in the presence of triethylamine, and the resulting product is purified chromatographically. Yield: 9 mg of blue lyophilizate (8195 from theoretical).

Elemental analysis: Calculated: C 51.21; H4.47; M4.88; 511.16; Ma 6.00

Found: C 51.50; H 4.55; M 4.95; 510.93; Ma 6.15

Example 14: Trisodium 3,3-dimethyl-2-(2-(1-II3,3-dimethyl-5-sulfonate-1-(2-sulfonatethyl)-ZH-indolium-2-yl|-vinylene)-2- 14-(2-16-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ugexylcarbamoylethyl)-phenoxy|cyclohex-1-en-3-ylidene)ethylidene1-1-(2- sulfonatoethyl)-2,3-dihydro-1H-indole-5-sulfonate, inner salt (formula XM) aa

M

Shchi / o (in; o more 5 52 (in) in o

AKA ANNA in Oh o-8-0 O-v-o Ma and I ma

The synthesis is carried out similarly to the poison example from 0.7 g (0.68 mmol) of the compound named in the title of example 14a and 0.53 g (1.22 mmol) of M-(13-amino-4,7,10-trioxatridecyl)maleimide-trifluoroacetate . Yield: 0.56 g of blue lyophilizate (6895 from theoretical).

Elemental analysis: Calculated: C 48.27; H 4.53; M 5.36; 5 12.27; Ma 6.60

Found: C 48.01; H 4.44; M 5.56; 512.10; Ma 6.81

Example 15: Trisodium 3,3-dimethyl-2-(2-(1-13,3-dimethyl-5-sulfonate-1-(2-sulfonatoethyl)-ZH-indolium-2-yl|vinylene)-2-( 4-(2-(113-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-4,7,10-trioxatridecylcarbamoylethyl)phenyxy|cyclohex-1-ene-Z-ylidene )ethylidene|-1-(2-sulfonatoethyl)-2,3-dihydro-1H-indole-5-sulfonate, inner salt (formula KhMI)

(04 in ote iv) oh oh oh oh! : ma (9) o

The synthesis is carried out similarly to the poison example from 0.7 g (0.68 mmol) of the compound named in the title of example 14a and 0.59 (1.3 mmol) M-(13-amino-4,7,10-trioxatridecyl)maleimide-trifluoroacetate . Two chromatographic purifications are carried out. Yield: 0.67g of blue lyophilizate (7595 from theoretical).

Elemental analysis: Calculated: C 52.29; H 5.16; M 4.28; 5 9.79; Ma 5.27

Found: C 51.88; H 5.40; M 4.34; 5 9.53; Ma 5.68

Example 16: Trisodium 3,3-dimethyl-2-(2-(1-(I3,3-dimethyl-5-sulfonate-1-(2-sulfonatoethyl)-ZH-indolium-2-ilyvinylene)-2-I4- (2-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-ethylcarbamoylethyluphenoxy|-5-tert.-butyl-cyclohex-1-en-3-ylidene) ethylidene|-1-(2-sulfonatoethyl)-2,3-dihydro-1H-indole-β5-sulfonate, internal salt (formula KHMII)

We ) / (g)

And o (9) t o vo c) ; o vo ay - t ma, o-v-o o-8-0 o

Ge) o a) M-((3-(Anilinomethylene)-2-chloro-5-tert.-butyl-1-cyclohexen-1-yl)umethyleneaniline hydrochloride 6b.7ml (73.4mmol) of phosphorus oxychloride is added dropwise at 0"C to 3ml of DMF. Then a solution of 5.0g (32.4 mmol) of 4-tert.-butylcyclohexanone in 30ml of dichloromethane is added dropwise, and the reaction mixture is stirred while heating under reflux for 10 hours. After cooling to 0"C, bg (64.9 mmol) of aniline in 5.5 ml of ethanol is slowly added dropwise, the mixture is transferred to 200 g of ice and, with stirring, 5 ml of concentrated hydrochloric acid is added. The solid substance that has precipitated is filtered off, washed with ether and dried. Yield: b .8 g (5095 from the theoretical) of a red solid substance. b) Trisodium 3,3-dimethyl-2-|2-(1-|13,3-dimethyl-5-sulfonate-1-(2-sulfonatoethyl)-ZH-indolium -2-yl|vinylene)-2-chloro-5-tert.-butyl-cyclohex-1-en-3-ylidene)ethylidene|-1-(2-sulfonatoethyl)-2,3-dihydro-1H-indole -5- sulfonate, inner salt 5.0 g (14.4 mmol) 1-(2-sulfonatoethyl)-2,3,3-trimethyl-ZN-indolenin-5-s sulfonic acid (example Ta) and 3.0 g (7.2 mmol) of M-(3-(anilinomethylene)-2-chloro-5-tert.-butyl-1-cyclohexen-1-yl)umethyleneaniline hydrochloride (example 16ba) are heated with under reflux together with 2.5 g (ZOmmol) of anhydrous sodium acetate in 100 ml of methanol for 1.5 hours, cooled, mixed with 150 ml of diethyl ether and stirred overnight. The precipitate is suctioned off, dried and purified by chromatography (silica gel, gradient: dichloromethane/methanol). Yield: 4.7 g of blue solid (6895 from theoretical). c) Trisodium 3,3-dimethyl-2-|2-(1-| 2-carboxyethyl)phenoxy!1-5-tert.-butyl-cyclohex-1-en-3-ylidene)ethylidene|-1--2-sulfonatoethyl)-2,3-dihydro-1H-indole-5-sulfonate, inner salt

The reaction is carried out with 2.0 g (2.1 mmol) of the compound named in the title of example 166, as described in example 136. Yield: 1.5 g (6690 from theoretical). d) Trisodium 3,3-dimethyl-2-|(2-(1-| (4-(2-Х2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-ethylcarbamoylethyl)phenoxy|-5-tert.-butyl-cyclohex-1-en-3- ylidene)ethylidene|-1-(2-sulfonatoethyl)-2,3-dihydro-1H-indole-5-sulfonate, inner salt

The reaction is carried out with 1.0 g (0.92 mmol) of the compound named in the title of example 16 as described in example 138. Chromatographic purification is carried out twice using AP-C18 silica gel (mobile phase acetonitrile/water). Yield: 0.24g (2295 from theoretical).

Elemental analysis: Calculated: C 52.82; H 4.93; M 4.65; 5 10.64; Ma 5.72

Found: С 52,23; H 5.20; M 4.31; 510.30; Ma 6.15

Examples 17-19: Synthesis of indotricarbocyanine dyes with bromoacetylamide groups

Example 17: Trisodium 3,3-dimethyl-2-(7-3,3-dimethyl-5-sulfonato-1-(2-sulfonatoethyl)-3H-indolium-2-yl|-4-(5-C6-( bromoacetylamino)hexylcarbamoyl|-4-oxapentyl)hepta-2,4,b-trien-1-ylidene)-1-(2-sulfonatoethyl)-2,3-dihydro-1H-indole-5-sulfonate, inner salt ( formula XIX)

about n

Bricklayer o o o (o) (c) ) o

Shchi m Oh m F

This is a) Trisodium 3,3-dimethyl-2-(17-1,3-dimethyl-5-sulfonato-1-(2-sulfonatoethyl)-ZH-indolium-2-yl|-4-(5 -(6-aminohexylhexyl)carbamoyl)-4-oxapentyl)hepta-2,4,b-trien-1-ylidene)-1-(2-sulfonatoethyl)-2,3-dihydro-1H-indole-5-sulfonate , internal salt

The synthesis is carried out similarly to example Td with 0.5 g (0.55 mmol) of the compound named in the title of example 10 and 0.15 g (0.70 mmol) of M-ros-hexanediamine (Rishka). The reaction product is purified using chromatography (AP

Sib5-chromatography, methanol/water gradient) and after lyophilization, mix under cooling with ice in a mixture of 2 ml of trifluoroacetic acid/dml of dichloromethane for 15 minutes. After centrifugation in vacuum, the residue is dissolved in methanol, precipitated with diethyl ether and isolated. Yield: 0.26g of blue solid (2295 from theoretical). b) Three sodium 3,3-dimethyl-2-(7-3,3-dimethyl-5-sulfonato-1-(2-sulfonatoethyl)-ZH-indolium-2-yl|-4-(5-(6- (bromoacetylamino)hexylcarbamoyl)-4-oxapentyl)hepta-2,4,b-trien-1-ylidene)-1-(2-sulfonatoethyl)-2,3-dihydro-1H-indole-5-sulfonate, internal salt 0 26g (0.23mmol) of the compound named in the title of example 18a is cooled in 3ml of DMF to -20"С, mixed with 2d3mg (0.28mmol) of triethylamine and a solution of 0.10g (0.46mmol) of bromoacetyl bromide in 0.2ml of dimethylformamide.

After stirring for 5 hours. at a maximum temperature of 0°C, precipitate by adding diethyl ether and obtain the product after reprecipitation from a mixture of dimethylformamide/diethyl ether and drying.

Yield: 0.23g of blue solid (8695 from theoretical).

Elemental analysis: Calculated: C 45.63; H 4.87; M 4.84; 5 11.07; Ma 5.96

Found: С 45,13; H 4.66; M 4.67; 5 10.83; Ma was not determined

Example 18: Trisodium 3,3-dimethyl-2-(7-3,3-dimethyl-5-sulfonato-1-(2-sulfonatoethyl)-ZH-indolium-2-yl|-4-(3-I3-( bromoacetylamino)propyl|carbamoyl)-ethyl)hepta-2,4,b-trien-1-ylidene-1-(2-sulfonatoethyl)-2,3-dihydro-1H-indole-5-sulfonate, inner salt (formula XM )

N kory z i ak i o o Y o this ih S. wa vy z Ach 9 y

And Yi cha. gu iz) 9

The synthesis is carried out from the compound named in the title of example 1g (0.5g; 0.5bmmol) and M-ros-propylenediamine similarly to example 17. Yield for all stages: 0.22g (3795 from theoretical).

Elemental analysis: Calculated: C 43.70; H 4.33; M 5.23; 5 11.96; Ma 5.96 5 Found: C 43.21; H 4.14; M 5.53; 5 10.89; Ma was not determined

Example 19: Trisodium 3,3-dimethyl-2-(2-(1-|3,3-dimethyl-5-sulfonato-1-(2-sulfonatoethyl)-ZH-indolium-2-yl|vinylene-2-( 4-I3-(bromoacetylamino)propylcarbamoyl)ethyl)phenoxy|cyclohex-1-en-3-Zylidene)ethylidene-1-(2-sulfonatoethyl)-2,3-dihydro-1H-indole-5-sulfonate, inner salt ( formula XX)

Nn N a a. о в) ох в оо тв (в) ЕЯ о » о (I M - F 7 4 M Fe

She t ma a -v-7 Ma o yo o i (in) ma

The synthesis is carried out from the compound named in the title of example 136 (0.5g; 049mmol) and M-ros-propylenediamine similarly to example 17. Yield for all stages: 0.31g (5395 from theoretical).

Elemental analysis: Calculated: C 47.88; H 4.52; M 4.65; 5 10.65; Ma 5.73

Found: C 48.04; H 4.43; M 4.69; 5 10.72; Ma 5.84

Examples 20-23: Synthesis of conjugates with biomolecules and photophysical characterization of conjugates

Example 20: Labeling of B5A (bovine serum albumin) with the compounds named in the titles of examples 1-16

General instructions: A solution of 5 mg (0.074 μmol) of BBA (Sotrapu bidta) in 5 ml of phosphate buffer (0.1 molar Mag?HPO4/MagPOYA4, pH 6.8) is mixed in each case with 0.74 μmol of the compounds named in the headings of examples 1 -16 (stock solutions for dilution of 0.5 mg/ml in PB5 (physiological solution with phosphate buffer), and incubated for 30 minutes at 25"C. Conjugates are purified using gel chromatography (column: Zerpadeh 250, diameter 1, 5cm, Rpaptasia, eluent: РВ5).

Example 21: Labeling of B5A with the compounds named in the titles of examples 17-19

General instructions: A solution of 5 mg (0.074 μmol) B5A (from Bidta Sotrapu) in 3 ml of phosphate buffer (0.1 M borate buffer, pH 8.5) is mixed in each case with 1.10 μmol of the compounds named in the titles of examples 17- 19 (stock solutions for dilution of 0.5 mg/ml in PB5), and incubated for 5 hours. at 25"C. Conjugates are purified using gel chromatography (column: Zerpadeh 250, diameter 1.5 cm, Riaptasia, eluent: РВ5).

Example 22: Labeling with SEM of ARZO antibody (fragment of a single chain) to ЕХО-В-fibronectin with the compounds named in the headings of examples 1-16.

ARZOU is zsEm with C-terminal cysteine and is present in the form of a 5-5-dimer with a molar mass of about 56,000 g/ml (Sy. Orip. Ogyd Oivzsom. YuOemei. 2002 Mag; 5(2): 204-13). During the reduction of disulfide bridges, two monomers with available 5H groups (molar mass 28 00oOg/ml) are formed.

General instructions: 0.3 ml of ARZ39 solution in РВ5 (concentration 0.93 mg of dimer/ml) is mixed with bomcl of tris(carboxyethyl)uphosphine (TCEP) solution in РВ5 (2.8 mg/ml) and incubated in a nitrogen atmosphere for 1 hour at 25760 .

Excess TSER is separated using gel filtration on a MAR-5 column (eluent: РВ5). The amount of the obtained ARZO monomer (OOgvony-1.4), determined by photometry, is 230-250 μg (volumes of 0.5-

Oh, bml). The solution is mixed with 0.0 μmol of the compounds named in the headings of examples 1-16 (starting solutions for dilution of 0.5 mg/ml in PB5), and incubated for 30 minutes at 25"C. The conjugate is purified using gel chromatography on MAR -5 columns (eluent: РВ5/1095 glycerol). The immunoreactivity of the conjugate solution is determined using affinity chromatography (EHUO-B-fibronectin resin) (U. Ittipoi. Meiy., 1999, 231, 239).

The immunoreactivity of the obtained conjugates is 28095 (ARZ39 before conjugation is 29595).

Example 23: Labeling with SEM of ARZO antibody (fragment of a single chain) to EO-B-fibronectin with the compounds named in the headings of examples 17-19

General instructions: 0.3 ml of ARZO solution in РВ5 (concentration 0.93 mg of dimer/ml) is mixed with 60 μl of tris(carboxyethylphosphine (TCEP) solution in РВ5 (2.8 mg/ml) and incubated in a nitrogen atmosphere for 1 hour at 25" Sat. The excess TSER is separated using gel filtration on a MAR-5 column (eluent: 5 mmol borate buffer, pH 8.5). The amount of the obtained ARZO monomer (Oyugvonm-1.4) determined by photometry is 230-250 μg ( volumes of 0.5-0.6 ml). The solution is mixed with 0.0 bm of the compounds named in the headings of examples 1-16 (starting solutions for dilution of 0.5 mg/ml in PB5), and incubated for 4 hours at 25" C. The conjugate is purified using gel chromatography on a MAR-5 column (eluent: РВ5/1095 glycerol).

The immunoreactivity of the conjugate solution is determined using affinity chromatography (EHUO-B-fibronectin resin) (9U. Ittipoi. Mei., 1999, 231, 239). The immunoreactivity of the obtained conjugates is 275956 (ARZ39 before conjugation 29590).

Photophysical characteristics of dye-B8BA conjugates of examples 21 and 22 and conjugates of dye-5cEm antibody of examples 23 and 24.

The degree of concentration (dye/antibody molar ratio) is determined photometrically, based on the extinction coefficient of 75,000 ml'/cm-! in the short-wave shoulder of the spectral line (about 690-710 Ohm); Absorption of antibodies (ARZ39) is determined with OYugvonm--1.4; and/or protein absorption (B5A) is determined with ОХгттнм -0.58.

The fluorescence quantum yield is determined with the help of 5PEK Piogoiyud (wavelength-dependent sensitivity is calibrated by the lamp and detector) relative to indocyanine green (0-0.13 in DMSO, 9U.Spet.

Ep. Oaiia, 1977, 22, 379, Viosopitsdage Spet., 2001, 12, 44).

Table 2

Properties of conjugates according to the invention

Degree of Absorption Fluores - Fluores

Substance (biomolecule/compound sample) concern cent cent cent tration maximum | maximum quantum pt (nm) yield oK2|Conjugate VZA of the compound named in the title applies | 06 | 77/67 | 7/9 | 016 (

K1'|Conjugatz B5A of the compound named in the title of example 4 | 0.8 | 767 | 792 | 02

K2I Conjugates VZA compounds named in the title of example 5 | 09 | /68 | 7/9 | 014

K16|Conjugatz VZA and compound named in the title of the example! | ID | 767 | 789.4 | 015 (

K17|Conjugatz B5A of the compound named in the title of example 2 | 09 | 766 | 790 | ol (k19|Conjugatz B5A of the compound named in the title of the application4 | 1,1 | 772 | 796 | 009

K21|VZA conjugates of the compounds named in the heading apply9 | 08| 773 | 754 | 03

Example 24:

Visualization properties of the conjugates according to the invention are investigated under the conditions of ip mimo after the injection of a hairless mouse that has a tumor. For this purpose, the conjugates are administered intravenously and the concentration in the area of the tumor is monitored for 24 hours. after the introduction. Fluorescence of substances is excited in the area of animal irradiation with the help of near-infrared light with a wavelength of 740O0nm, which is obtained with the help of a diode laser (0.5UU output). Fluorescent radiation is detected using an enhanced SSO camera, and fluorescent images are stored digitally. The effectiveness in the conditions of ip mimo dye conjugates is shown in Fig. 2, based on an example.

It may be believed that any person skilled in the art may, without further elaboration, utilize the present invention to its fullest extent using the foregoing description. Therefore, the following preferred specific variants should be interpreted only as illustrative and as not limiting in any way the other scope of the invention.

The complete description(s) of all applications, patents and publications cited herein are incorporated herein by reference.

The preceding examples may be repeated with similar success by substituting the general or specifically described regents or technical terms of the present invention for their use in the preceding examples.

From the foregoing description, any person skilled in the art can readily ascertain the essential characteristics of the present invention and, without departing from its spirit and scope, can make various changes and modifications to the invention to adapt it to various applications and conditions.

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Fig.

Claims (1)

1. Indotricarbocyanine dye of the formula (I) Kk Deu M Ko Kk, M ! ! I Kk, ; (0) where X means a doubly substituted carbon atom, while the substituents are selected from methyl, ethyl, propyl, isopropyl and/or butyl groups, U means CH»АСН» or СНОАСНО-СН» 7. means C-alkyl, while the carbon atoms are optionally substituted by O or 5, or are o. 2 Ki-Ka independently of each other mean 5OzH or H, provided that at least three of Ki-K/a mean 5ОЗН, K5 means -СО-МН-Ка-К»о, -МН-С5-МН-К» -Co or -МН-СО-Ка»-Ко, in which Ku» is selected from the group consisting of unbranched C0 isalkyl, in which the C atoms are optionally substituted by O or 5.1 Co selected from о -th is) ; -МН и: (c) or chloroacetyl, bromoacetyl, iodoacetyl, chloroacetamido-, iodoacetamido-, chloroalkyl, bromoalkyl and iodoalkyl, and in which Kb and K7 mean CH or C, connected to each other through a trimethylene link, which is not required can necessarily be substituted by Silalkyl, with the formation of a six-membered ring optionally substituted in the y-position by Silalkyl, or a salt and solvate of this compound. 2. Indotricarbocyanine dye according to claim 1, which differs in that U means СНо-СН», 7 means Si-alkyl, while the carbon atoms are optionally replaced by O or 5, and in which Ke and K7 mean CH, 1 salts and solvates of this compound. 3. Indotricarbocyanine dye according to claim 1 or 2, which differs in that 7 means Si-alkyl. 4. Indotricarbocyanine dye according to claim 1 or 2, which differs in that 7 stands for o. and Kb and K7 each mean C atoms connected to each other through a trimethylene link to form a six-membered ring. 5. Indotricarbocyanine dye according to claim 3, which differs in that it corresponds to the following formula (P) c) M more, o) MH o o, r o von DA AC MA OH OH (1) or salts and solvates of this compound. 6. Indotricarbocyanine dye according to claim 3, which differs in that it corresponds to the following formula (ПШ) р (c) М (c) МН ул о r КЗ ОН ПОДА А АЙУ МА ОН ОН (FP) or salts and solvates of this compound. 7. Indotricarbocyanine dye according to claim 3, which differs in that it corresponds to the following formula (IM) (o) c) MH (c) c) ml lys lych Y oh r he von c) (c) Di MA OH zOZN (M ) or salts and solvates of this compound. 8. Indotricarbocyanine dye according to claim 3, which differs in that it corresponds to the following formula (U) (o) Ox MH o (c) o, /9 Z) von PODA Me A ZON ZON (U) or salts and solvates of this compound. 9. Indotricarbocyanine dye according to claim 3, which differs in that it corresponds to the following formula (MI) (o) a a a Mi (c) oX ,/9 7 von POD iS M A OH OH «UU or salts and solvates of this compound. 10. Indotricarbocyanine dye according to claim 3, which differs in that it corresponds to the following formula (UP) (o) (c) MH c) (c) M (c) o, r ZON (c) Di M" A OH OH ( lu or salts and solvates of this compound. 11. Indotricarbocyanine dye according to claim 3, which differs in that it corresponds to the following formula (М Ш) (o) He) MH (c) o- OH and in on (c) oka MA von OH (UP or salts and solvates of this compound. 12. Indotricarbocyanine dye according to claim 3, which differs in that it corresponds to the following formula (IX) (o) o MH and Mi (c) o-Ou and 58 von PDAA AAT MA OH OH (u or salts and solvates of this compound. 13. Indotricarbocyanine dye according to claim 3, which differs in that it corresponds to the following formula (X) (o) (c) MH c) (c) M zhi eta il i (c) ОХ r 7 won sho ov M A won won su or salts and solvates of this compound. 14. Indotricarbocyanine dye according to claim 2, which differs in that it corresponds to the following formula (XI) (o) (c) M o o o- Ou i von (c) DAO MA von von c) or salts and solvates of this compound. 15. Indotricarbocyanine dye according to claim 2, which differs in that it corresponds to the following formula (XP) (o) (c) ул А, о о о- Оу и о ОН DAO MA вОЗН von Оса) or salts and solvates of this compound. 16. Indotricarbocyanine dye according to claim 2, which differs in that it corresponds to the following formula: ) or salts and solvates of this compound. 17. Indotricarbocyanine dye according to claim 4, which differs in that it corresponds to the following formula (CM) (c) Mn and I (c) (c) ОХ r KZ iv) on PO M A OH ОН «CaU) or salts and solvates of this compound. 18. Indotricarbocyanine dye according to claim 4, which differs in that it corresponds to the following formula (XU) (o); M and Mi (c) (c) ohri KZ o on oven M" A ON OZN SU) or salts and solvates of this compound. 19. Indotricarbocyanine dye according to claim 4, which is characterized by the fact that it corresponds to the following formula (XM) (o) M y tatics o o o- Ox i 58 o OH o Y orav CO A OH OH U or salts and solvates of this compound. 20. Indotricarbocyanine dye according to claim 4, which differs in that it corresponds to the following formula (HMP) (c) MH yam (c) (c) o-Ox, 58 o won (c) SD SO A won won XM) or salts and solvates of this compound. 21. Indotricarbocyanine dye according to claim 3, which differs in that it corresponds to the following formula (XM) Mn (I is He) МН is) o- ОХ and 58 von PO AA Me A OH ОН z З ХУ) or salts and solvates of this compound . 22. Indotricarbocyanine dye according to claim 2, which differs in that it corresponds to the following formula (XIX) c) лу МН те о о о- Оу и в вон (c) ДАТ МА ОН ОН сг) or salts and solvates of this compound. 23. Indotricarbocyanine dye according to claim 4, which differs in that it corresponds to the following formula (XX) INN uv (c) (c) o- ee o von pOBADAKO ee OH on su or salts and solvates of this compound. 24. Compound of formula (I) Kk, A Kk x 7 X I Du M Ko Kk, M ! ! к Кк, , () in which X means a doubly substituted carbon atom, while the substituents are selected from methyl, ethyl, propyl, isopropyl and/or butyl groups, U means CH»АСН» or СНОАСНО-CH», 7 means Sisalkyl, when its carbon atoms are optionally substituted by O or 5, or is an oxyphenyl fragment of o. 2 Ki-Ka independently of each other are 5OzH or H, provided that at least three of Ki-K/a are 5OZN, Co is -COOH, and Co and K7 are CH or C linked together through a trimethylene link , which can optionally be substituted by C-alkyl, with the formation of a six-membered ring optionally substituted in the n-position by C-alkyl, or a salt and solvate of this compound. 25. The method of obtaining an indotricarbocyanine dye according to any of claims 1-16 or 21-23, which has the following general formula (1) i Kk, ,; 0, which differs in that it includes the following stages: a) obtaining an indolinine derivative, b) preparation of one or more 4-substituted pyridines, c) conversion of one or more 4-substituted pyridines into meso-substituted glutaconaldehyde dianilides using the Zincke-Koenig reaction, 1 d) interaction of the compound obtained in stage a) with the compound obtained in stages c), and e) introduction of fragment 7-K5 to the compound obtained in stage d). 26. The method of obtaining conjugates, which is characterized by the fact that it includes the addition of an indotricarbocyanine dye according to any of claims 1-23 to a biomolecule containing a 5H group. 27. Conjugate of an indotricarbocyanine dye with a biomolecule, which differs from that obtained according to claim 26. 28. The conjugate according to claim 27, which is characterized in that the biomolecule includes at least one biomolecule selected from peptides, proteins, lipoproteins, antibodies or antibody fragments, nucleic acid, such as, for example, oligo- or polynucleotides from DNA or RNA, aptamers and RNA. 29. The conjugate according to claim 28, which is characterized by the fact that the protein is selected from the group of structural proteins or soluble proteins of the body. 30. Conjugate according to claims 28 or 29, characterized in that the soluble protein is a serum protein, such as, for example, H5A, B5A, egg albumin, an enzyme, such as, for example, peroxidase, or an antibody, a 5cEu fragment or K (ab). 31. The conjugate according to any of claims 27-30, which is characterized in that the soluble protein has an affinity for EO-B-fibronectin. 32. The conjugate according to any one of claims 27-31, which differs in that the indotricarbocyanine dye is attached to the biomolecule through a 5H group, in particular through a 5H group to cysteine. 33. A diagnostic kit, which is characterized by the fact that it includes an indotricarbocyanine dye according to any of claims 1-23 and/or a biomolecule containing a 5H group, together with additional adjuvants for carrying out diagnostics under the conditions of il ui»o, in particular tumors 34. Use of the conjugate according to any one of claims 27-31 as a fluorescent contrast medium for the diagnosis of tumors.