PT92487B - PROCESS FOR THE PREPARATION OF PEPTID DERIVATIVES - Google Patents

Abstract

Somatostatin peptides bearing at least one chelating group for a detectable element, said chelating group being linked to an amino group of said peptide, and said amino group having no significant binding affinity for somatostatin receptors, in free or salt form, are complexed with a detectable element and as such are useful as a pharmaceutical, e.g. a radiopharmaceutical for in vivo imaging of somatostatin receptor positive tumors or for therapy.

Description

The present invention relates to a process for the preparation of polypeptides, the pharmaceutical compositions containing them and their use as pharmaceuticals, for example for the treatment of somatostatin receptor positive tumors for live diagnosis providing images' of the agents.

I

In recent years there has been a high incidence of somatostatin rheepores in several human tumors, for example tumors! pituitary, central nervous system tumors, breast tumors,? gastro-enteropancreatic tumors and their metastases. Some of them are already small or slow growing tumors that are difficult to pinpoint with conventional diagnostic procedures

The in vitro visualization of somatostatin receptors has been re-performed through autoradiography of tumor tissues using iodized somatostatin or somatostatin analogs, for example £ ¹² 5l-Tyr ¹ ^ 2-somatostatin-14 (Taylor, JE et al., Life Science (198S) 43 »421), or, f ¹²⁵ I-Tyr ³ / SMS 201-995 also called / ¹² \ / 204-090 (Reubi, JC et al., Brain Res. (1987) 406: 89I; Reubi , JC et al., J.Clin. Endocr. Metab. (1987) 65; 1127; Reubi, JC et al., Cancer Res. (1987) 42 /: 551} JC et al., Cancen Res. (1987) 42. »5758).

New somatostatin peptides that have been found to be therapeutically useful and can be labeled for therapeutic and in vivo diagnostic applications have now been discovered.

According to the invention, a somatostatin peptide is developed which has at least one chelating group of a detectable element; said chelating group being attached to an amino group of said peptide, said amino group having no significant binding affinity with respect to somatostatin receptors. ί

I

These compounds are hereinafter referred to as DA LIGANDS. INVENTION. They have a chelating group capable of reacting with a detectable element, for example a radio-nucleated element, a radio-opaque element or a paramagnetic ion, to prepare a complex: and subsequently be able to bind to the summation receptors.

tostatin, for example expressed or over-expressed by tumors or metastases.

The chelating group is linked to the amino group of the peptide by a covalent bond. The chelating group is preferably linked to the N-amino group

I terminal of the somatostatin peptide. j j

According to the invention, the chelating group can be linked either directly or indirectly, for example by means of an inter-group. median to the amino group of the somatostatin peptide. j

A group of LIGANDS is one in which the chelating group is linked directly; the amino group of the somatostatin peptide.

Another group of ligands is one in which the chelating group is linked indirectly by an intermediate group. bridge, to the amino group of the somatostatin peptide.

Preferably the linker group is linked by an amide bond to the peptide.

The term "somatostatin peptide includes naturally occurring soaatostatins (tetradecapeptide) and their analogs or derivatives.

The terms "analogues or derivatives" as used herein, mean any linear or cyclic chain polypeptide, derived from the naturally occurring somatostatin tetradecapeptide, in which one or more amino acid units have been removed and / or replaced by one or more radicals of other amino acids and / or in which one or more functional groups have been replaced by one or more other functional groups and / or one or more groups have been replaced by one or more other isosteric groups. Generally, the term embraces all de-!

modified derivatives of a biologically active peptide, which has a qualitatively similar effect to that of the unmodified somatostatin peptide, for example they bind to somatostatin receptors and decrease hormonal secretion.

The analogues of straight chain somatostatin, cyclic bridged and cyclic | clinicians are known compounds. Such compounds and their preparation are described, for example, in European Patent Specification I ep-a-1295; 29.5795 215.1715 203.031; 214,872; 298,732; 277,419.

The preferred LIGANDS of the invention are those derived from the following somatostatin analogs:

A. Formula I analogs

THE

ch ₂ -sy ₁ y ₂ -s-ch ₂ n-ch-cO-BCDE-NH-CH-G (I) in which the symbol A represents C7-C10 alkyl or a group of

C ^ -C ^ ₂ , phenyl-alkyl in formula RCO-, where

i) osimbolo R represents hydrogen, alkyl in

0 ^ -0 ^, phenyl or phenyl-alkyl in ^or ii) RCO- e

a) an L- or D-phenylalanine residue optionally ring - substituted by F, Cl, Br, Nog, NHG, OH, C ^^ - ^ C and / or alkoxy C ₁ -C +;

b) the residue of a natural or synthetic a-amino acid, different from that previously defined in a), or of a corresponding D-amino acid, or

c) a dipeptide residue, where the individual amino acid residues are the same or different and are chosen from those defined in a) and / or b)

βί-amino group of the amino acid residues a) and h) and the N-amino terminal group of the dipeptide residues c) being optionally monoI or di-alkylated at ^or P ^or alkanoyl in ί simhola A · ^Y 1 ^{and Y} 2 represents hydrogen, C ^-C. alkyl ^or phenyl-alkyl together represent a direct bond or each of the halo Y ^ and Y ^ are, independently of each other, hydrogen or a radical of the

formulas (l) to (5) R CH _n The I 1 -CO-C- (CH ₂ ) _m -H I -CO-CH 1 ^R h (I) (2)

-CO-NHR -CO-NH-CH-COOR ç 1 ⁰ R. (3) d (4) R, “ The* -CO- (NH) _p - Ç - ^(0H ^ -O ^ ⁸⁸ R., £> ’> \ _R S!

in which (5) ^R bm

^R a ' ^{and R} b' ^r 8 ^{and R} 9

Q q

Γ

B is methyl or ethyl is hydrogen, methyl or ethyl!

is an integer from 1 to 4!

i is an integer from 1 to 5 is C1 -Cg alkyl represents the substituent attached to the carbon atom of a natural or synthetic cf-amino acid (including; hydrogen);

is C-C _r alkyl

5 'are independently of each other, hydrogen, methyl or ethyl, are independently of each other hydrogen, halogen, C1 -C4 alkyl or C4 -C4 alkoxy; is 0 or 1 is 0 or 1, and is 0 | 1 or 2, is Phe-optionally substituted on the ring with a halogen, NC> 2 NH ₂ , OH, C ^ -C ^ alkyl and / or C ^ -C ^ alkoxy (including pentabluror-alanine), or ^ - naphthyl-Ala;

C is (L) -Trp- or (D) -Trp- optionally <(- N-methylated and optionally substituted on the benzene ring with a halogen,

NO ^, NH ^, OH, C ^ -C ^ alkyl and / or C ^ -C ^ j alkoxy

D is Lys, in which the side chain comprises an atom of 0 or S in the Ji position, tF-Lys or oF-Lys, optionally o-methylated, or a 4-amino-cyclohexyl-Alaj residue

I or a 4-amino-cyclohexyl-Gly is Thr, Ser, Val, Phe, Ile or a residue of araib non-isobutyl or aminobutyric acid is a group of formula in which

-coor ₇ , -ch ₂ or _1o ,

is hydrogen or C1 -C4 alkyl is hydrogen or a physiologically acceptable, physiologically hydrolyzable residue;

is hydrogen, C C-C ^ alkyl, phenyl or phenylalkyl in θγ- ^ θ;

is hydrogen, alkyl in or a group of formula

-ch (r ₁₃ ) -x ₁ , is CH ₂ OH, - (CH ₂ ) ₂ -OH, - (CH ₂ ) ₃ -OH, or -CH (CH ₃ ) OR or represents the substitutent attached to the carbon atom in the position of a c (-natural or synthetic amino acid (including hydrogen) and is a group of the formula -COOR ?; -Cí ^ OR ^ q or in which ^R 7 ® ^R 10

R.

R.

have the meanings mentioned above;

is hydrogen or C ^ -C ₃ is hydrogen, C ^ -C ₃ alkyl , phenyl or fenil14 '15. ¾-

ί-alkyl in Ογ-Ο ^ θ, and is hydrogen or hydroxy, with the proviso that when R ^ ₂ is -CH (R ^^) - X ^ then R ^ j is hydrogen or methyl,: in which the residues B, D and E have the L configuration, and the residues at positions 2 and 7 ® any of the residues Y ^ 4) θ 4) each have 'independently the (L) or (d) configuration.

The meanings of A and A 'in formula I are preferably chosen such that the compound comprises an -NH- terminal group capable of binding to a chelating agent.

In the compounds of formula I, the following meanings are preferred, both individually, and in any combination or sub-combination:

1. The symbol A represents: phenyl-alkyl, especially phenyl-ethyl, or a group of formula RCO. Preferably, symbol A represents a group of formula RCO.

1.1 Preferably the symbol R represents a C ^ -C ^^ alkyl ^or a phenyl-alkyl in θγ “θιθ” ^es P ^ec -'- ^a T ^men 'te & a phenyl-alkyl in C ^ -C ^ q, preferably it is phenylethyl, or a group of formula RCO has the meaning a), b) or c).

1.2 When RCO means a), b) or c), the group c (-amino of the amino acid residues a) and b) and the N-terminal amino group of the dipeptide residues c) is preferably non-alkyl or mono-alkyl in C ^ -C ^ ₂ P ^{s c} i l te ^men alkylated C ^ -C, n> i ^are especially methylated. Preferably, the N-terminal group is not alkylated. ;

1.3 When the group of formula RCO has the meaning described in a) it is preferably a) a L- or D-phenyl-alanine or - tyrosine residue, preferably mono-N-alkylated at C ^ -C ^ ₂ · preferably a ') is an L- or D-phenyl-alanine residue.

1.4 .5

When the group of formula RCO has the meaning referred to in b) or c), the residue referred to is preferably lyophilic. Preferred residues b) are therefore those defined in b ') residues c (-amino acids having a hydrocarbon side chain, for example alkyl with 3, preferably 4 or more carbon atoms, for example up to 7 carbon atoms, naphthyl-methyl or hetero-aryl, for example 3— (2— or 1-naphthyl) -alarine, pyridyl-methyl or tritophane residue, having the residues cited configuration L-or D-, and residues i

preferred in C) preferred are dipeptide residues in which individual amino acid residues are identical or different [

Irent and are chosen from the e defined above in a *) and b ·) An example of a residue c) is 3- (2-naphthyl) -alanine residue.

group of the most preferred RCO formula has the meaning · a) especially a *)

B and B ', where B' is PHe or Tyr.

C and C ', where C' is (D) Trp.

DéB ', where D' is Lys, Melys or Lys (E-Me), especially Lys.

E and E ’, where E * is Vai or Thr, especially Thr

G and G ’, where G 'is a formula group - CO-N

especially a group of formula -CO-N ch (r ₁₃ ) -x ₁ .Λ, 7 Õ * (in which case R ^ j ^ or CH ^). In the latter case the structure -ChÇr ^^) - -X ^ preferably has the L configuration.

6.1

6.2 * 11 ^€ preferably hydrogen.

As a substituent attached to the carbon atom at the position of a natural amino acid (i.e., formula H ^ N-CH (r ₁₃ ) -COOH), R ₁₃ is preferably -CH ₂ -OH, -CH (CH ₃ ) -OH, isobutyl or butyl, or R ₁₃ is (CH ₂ ) ₂ -OH or - (CH2 ^ -OH.

$ especially -CH ₂ OH or -CH (CH,) OH.

6.3

X ^ is preferably a group of the formula -CO-N or -CH ₂ -CR ^ q, especially of the formula -ΟΙ ^ -ΟΑ ^ θ, * 15 ^and * 10 is preferably hydrogen or has the meaning given below in point 7. Most preferably R ^ q is hydrogen

The following individual compounds are illustrative of the compounds of formula I:

H- (D) Phe-Cys-Fhe- (D) Trp-Lys-Thr-Cys-Thr-ol also known as octreotíó-ôo (D) Phe-Cys-Thr- (D) Trp-Lys-Val-Cys -ThrNH ₂ l

(D) Phe-Cys-Tyr- (D) Trp-Lys-Val-Cys-TrpNI ^ (D) -Trp-Cys-Phe- (B) Trp-Lys-Thr-Cys-ThrWI ^ (D) -Phe -Cys-Phe- (D) Trp-Lys-Thr-Cys-ThrNH ₂ ^Γ 11

I-1

3- (2-naphthyl) - (D) Ala-Cys-Tyr- (D) Trp-Lys-Val-C ys-ThrNH _r

3- (2-naphthyl) - (D) Ala-Cys-Tyr- (D) Trp-Lys-Val-Cys- ^ 3-Nal-NH „

3- (2-naphthyl) - (d) Ala-Cys-B-Nal- (D) Trp-Lys-Val-Cys-TbrNH ^ (D) Phe-Cys-Phe- (D) -Trp-Lys-Thr -Cys-B-Nal-NH _r

B. Analogs of formula II

I-1

H-Cys-Phe-Phe- (D) -Trp-Lys-Thr-Phe-Cys-ol II / see Vale et al., Metabolism, 27, Supp. 1, 139, (1978) /

I-1

H-C ys-Hi s-Hi s-Phe-Phe- (D) Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH III (see EP-A-2OO, 188)

The contents of all previous publications, including the specific compounds, are specifically incorporated herein by reference.

Particularly preferred LIGANDS are those derived from:

H- (D) Phe-Cys-Phe- (D) Trp-Lys-Thr-Cys-Thr-ol

Suitable chelating groups are physiologically acceptable chelating groups, capable of complexing a detectable element. j

Preferably the chelating group has substantially a hydroxylic character. Examples of chelating groups include amino-dicarboxylic groups, polyamino-polycarboxylic groups, for example those derived from non-cyclic ligands, for example ethylene diamino acid12

-tetra-acetic (SDTA), diethylene triamine penta-acetic acid (DTPA), ethylene glycol-0-0'-bis- (2-amino-ethyl) -N-, N, N *, H'-tetra- acetic (EGTA), N, N * -bis (hydroxybenzyl) ethylenediamine-N-N'-diacetic acid (HBBD) and hexa-acetic triethylene tetraamine (TTHA), the derivatives;

!

! substituted EDTA or DTPA, for example p-isothiocyanate-benzyl-EDTA; or —DTPA, the derivatives of macrocyclic ligands, for example 1,4,7,10-tetra-azacyclo-dodecan-N, N, N, N'-tetra-acetic acid (DOTA) and 1,4,8, ll-tetra-azacyclododecane-N, N ^f , N ',' N - ”'_- fetra-acetic (TETA) derivatives of N-substituted or C-substituted macrocyclic amines', including also scylamates, for example Euro-European patent 304,780 AI and WO 89 / OI476-A, groups of formula IV or V

0 0 | II II

Hj -OS- (CH ₂ ) _n , -C- (TTj.-O- _IV i!

R ₂ -CS- (CH ₂ ) _n , -G -NH-CH,

CH-C _R CS- (GH) - CN ³ II íl in which each of the symbols R ^, R ₂ and R ^ are independently C ^ -Gg alkyl, C-Cg aryl or C-aryl alkyl - G ^, each optionally substituted by OH, G ^ -G ^ alkoxy, GOOH or SO ^ H, is 1 or 2 i is an integer between 2 and 6, and

TT are independently c (- or J3-amino acids linked to each other by amide bonds, groups derived from bis-aminothiol derivatives, for example compounds of formula VI is w »'

SE

HS

wherein each one of R _20> ^R 21 ^'R 22 ^{and R} 23 ^{are inde} P ^ ° ^{Enden' a3men 'te} each other hydrogen or C₁-C₄ alkyl, X ₂ is a group capable of reacting with an N-amino- group of the peptide, in * is 2 or 3>

group derived from di-thiasemicarbazone derivatives, for example compounds of formula VII

VII in which

Xj is the aforementioned one, groups derived from propylene amine oxime derivatives, for example compounds of formula VIII

1/

VIII in which each of the groups ^R 26 '^ 27 * ^ 28 ^and & 29 ^sa ° ^ ⁿ ^ - ^and P ^{en (} -'- ^en ' 1 ' ^emen ^ ^and each other hydrogen or C ^ -C ^ alkyl, and

X ₂ in are those already defined above; groups derived from dimercaptoid diamine _; for example compounds of formula IX

IX in which

X- is an optionally substituted divalent radical, which has a group capable of reacting with the N-amino group of the peptide, for example C ^-C ^ alkylene or phenylene which has an X ^ group, and is hydrogen or CO ₂ R ₃ q, in which R ^ _q is C ^-C ^ alkyl; groups derived from porphyrins, for example N-benzyl-5-10,15,20-tetrakis- (4 ^- carboxyphenyl) porphine, or TPP, which has an X ₂ group as defined above.

Aryl is preferably phenyl, aryl-alkyl is preferably benzyl.

Examples of X ^ include radicals of the formula -X ^, ^{in the}

X. is alkylene in C ^ -C ^ J ^ου · alkylene in C ^ -Gg optionally linked to:

oxygen atom or by -NH-;

í n * *:

is 0 or 1 and X4 is -NCS, a carboxy group or a functional derivative thereof, for example an acid halide, acid anhydride or acid hydrazide. It is understood that the group X4 is attached to a carbon atom of the ^0U group in place of a hydrogen atom. J j

chelating group can be linked, either directly or indirectly, to the N-amino group of the somatostatin peptide. When connected indirectly, it is preferably connected via a bridged group or a spacing group, for example a group of formula (<(1)

Z — R — C 0— (<1)

R ^ _c is an alkylene in C. -CL. , C-C. alkenylene, or -CH (R ') 35 1 11 2 11 in which R' is a residue attached at position c (to a natural or synthetic qf-amino acid, for example hydrogen, 'C-alkyl ^ -C ^ j, benzyl, optionally substituted benzyl, naphthyl-methyl, pyridyl-methyl

Z is a functional structure capable of reacting covalently with chelating agents.

Z can be, for example, a group that can form an ether, ester or amide bond with the chelating group. Z is preferably amino.

Chelating groups, when comprised of a carboxy group, may have -SO ^H and / or amino groups in the free or salt form.

Preferred chelating groups are those derived from polyamino-polycarboxylic groups, for example those derived from EDTA, DTPA, BOTA, TSTÂ, or substituted EDTA or DTPA. The most preferred chelating groups are

derivatives of DTPA.

In the LIGANDS OF THE INVENTION, the chelating group, when polyfunctional, can be linked to either a single molecule or somatostatin peptide, i

I like more than one, for example two molecules of the so- [

I matostatin. j

The LIGANDS OF THE INVENTION can exist for example in free or salt form. The salts include acid addition salts such as organic acids, polymeric acids or inorganic acids, for example hydrochlorides and acetates, and in the form of salts obtained with the | carboxylic or sulphenic acids present in the chelating group, for example

I alkali metal salts, such as sodium or potassium, or substituted or unsubstituted ammonium salts.

The present invention also includes a process for preparing the LIGANDS OF THE INVENTION, which can be produced by analogy with known methods.

The LIGANDS OF THE INVENTION can be prepared for example as follows i

a) removing at least one! in a somatostatin peptide, a protecting group, which is present that has a chelating group, or

b) by linking to each other, by means of an amide bond, two peptide fragments each containing at least one amino acid, or an amino alcohol in protected or unprotected form and one of them has the chelating group, and in which the amide is bonded in such a way as to obtain: the desired amino acid sequence, and then optionally, a) of the process, or

c) by binding to each other a chelating agent and the desired somatostatin peptide, in protected or unprotected form, so that the

chelating group is fixed in the desired N-amino group of the peptide, and then optionally a) is performed: ou / 7 β 'f //

d) removing a functional group from a protected or unprotected peptide, which has a chelating group or transforming that group to another functional group, in order to obtain another protected or unprotected peptide, which it has a chelating group and in this last case, the procedural operation a) is carried out; or i

e) oxidizing a modified somatostatin peptide with a chylating group, in which the mercapto groups of the Cys radicals are in free form, to produce an analog, in which the two Cys radicals are connected by an SS bridge and recover the LIGANDO thus obtained, in the free form or in the form of salt.

The above reactions can be carried out in analogy with known processes, for example as described in the examples that follow, particularly with processes a) and c). When the chelating group is linked by an amide bond, it can be carried out analogously to the processes used for the formation of amide. When desired, in these reactions the protecting groups, which are suitable for use in peptides or the desired chelating groups, can be used for functional groups that do not participate in the reaction. The term protecting group may also include a resin polymer that has functional groups.

When it is desired to link the chelating group to the terminal N-amino group of the peptide or peptide fragment used as the starting material, and which comprises one or more side chain amino groups, these are co-protected with a protective group, for example, as used in peptide chemistry nOnce _the desired turn on the chelating group on the side chain amino group of a peptide or peptide fragment used as starting material and that the peptide comprises an N-terminal amino group livi'e, this ! the latter will preferably be protected with a protecting group.

peptide fragment that supports the chelating group and that is used in phase b) can be prepared by reacting the peptide fragment that with — j i

it comprises at least one amino acid or an amino alcohol, has the protected or unprotected form with the chelating group. The reaction can be carried out in analogy to step c) ·

The chelating groups of formulas IV or V can be linked to a peptide by reacting * the chelating agent of formula IV * or V *

0 0 i ^ -csC ch ₂ ) _n , -G- (TT).-CX

IV »

R ₉ -CS- (CH ₂ ) _n , -C-NH — CH,

II

CH-C-X

R ₃ -CS- (CH ₂ ) _n , -C-NH

II II

0 in which X is an activating group capable of forming an amid bond. with the N-amino group of the peptide. The reaction can be carried out in accordance with European Patent 247 · 866 A1.

chelating agent used in step c) can be one already known, or be prepared in analogy with known techniques. The compound used is such that it allows the introduction of the desired chelating agent into the somatostatin peptide, for example a polyamino-polycarboxylic acid as disclosed, a salt or anhydride thereof.

In the previous process, when in amino acids, peptide fragments or peptides, used as a starting material, the kelaii group is connected via a bridge or a spacer group to the foot

For example, a radical of formula (cCl) as defined above, such amino acids, peptide fragments can be prepared by reacting in a conventional manner, the amino acids corresponding to the bridging group or a spacer group capable of forming a bridging or for example an acid or derivative reactio bridging or distancing group, by I

ZR ^ _c -COOH or a reactive ion derivative thereof. Examples of active ester groups or

Free peptides or peptides with a corresponding compound to separate the acid, which for example comprises an acid with an acid formula such as an ester with carboxy activating groups are, for example, κηρίο 4-nitro-phenyl, pentachlorophenyl, pentafluorphenyl succinimidyl or 1- hydroxy-benzotriazolyl.

í

Alternatively, the chelating agent can first react with a compound capable of forming the bridging or producing a distancing group, in order to support the bridging or distancing group and then react in a conventional manner with the peptide, the peptide fragment or the amino acid.

The LIGANDS OF THE INVENTION can be purified in a conventional manner, for example by chromatography. Preferably, the LIGANDS OF THE INVENTION comprise less than 5% ^by weight of peptides free of chelating agents.

The LIGANDS OF THE INVENTION in free form or pharmaceutically acceptable salts are valuable compounds. According to other embodiments, the LIGANDS OF THE INVENTION can be complexed with a detectable element.

Consequently, the present invention also develops the LIGANDS OF THE INVENTION as defined above, which are complexed with a detectable element (hereinafter referred to as CHELATES OF THE INVENTION), in the free form or in a salt, their preparation and the its use for therapeutic treatment and in vivo diagnostics.

Detectable element means any element, preferably [a metal ion that exhibits a detectable property in dl techniques

In vivo agnostics or therapies, for example a metal ion that emits detectable radiation, or a metal ion that is capable of influencing the NMR-lowering properties.

Suitable detectable metal ions include for example heavy elements or rare earth ions, for example those used in TAC (Computed Axial Tumography), paramagnetic ions, for example Gd ^J , Mn and Cr2 +, fluorescent metal ions, for example Eu ^, and radionuclear elements , for example radionuclei that emit gamma, beta i or alpha radiation and radioisotope elements that emit positrons, for example ⁶⁸ Ga. !

i

The radioisotope elements emitting gamma radiation include those that are useful in diagnostic techniques. Gamma-radiation radioisotopes advantageously have an average life span; between 1 hour and 40 days, preferably between 5! hours and 4 days, more preferably between 12 hours and 3 days. Examples are radio isotopes derived from gallium, fndium, technetium, Iterbium, rhenium, and thallium for example ^^ Ga, ^^ ^m Tc, l9Yb and '^ Re,

Of radioisotopes emitting gamma radiation are selective preferenciaos ¹ · tioned due to the metabolism INVENTION ligands or peptide somatostatin used. More preferably, the LIGAND OF THE INVENTION is chelated with a radioisotope-gamma that has a longer average lifespan than the average lifespan of the somatostatin peptide on the tumor.

Other suitable radioisotopes include those which emit positrons, for example, _the aforementioned

Suitable radioisotopes emitting beta radiation include those that are useful in therapeutic applications, for example ^ θΥ, ^ ”^ Cu, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁶ ? Er, ¹²¹ Sn, ^ Te, ¹⁴² Pr. Beta isotopes advantageously have an average life span of about 2.3 hours to 14.3 days, preferably between 2.3 and 100 hours. Beta radioisotopes are preferably chosen in the sense that they have a longer average life span than average life! day of the somatostatin peptide in the tumor.

Suitable alpha radioisotopes are those that are used to treat:

211 212 therapeutic instruments, for example At, Bi.

CHELATES OF THE INVENTION can be prepared by reacting the BINDING with a corresponding detectable element, giving the compound, for example a metal salt, preferably a water-soluble salt. The reaction can be carried out. carried out by analogy to known techniques, for example as disclosed by Perrin in Organic, Chemical Data Sereis, 22. NY Pergamon Press (1982); by Krejcarit and Tucker, in Biophys, Biochem, Res. Com. 77t 5θ1 (1977) and by Wagner and Welch, in J. Nucl. Med. 20: 428 (1979).

Preferably the ligand is complemented at a pH at which the

CONNECTING THE INVENTION is physiologically stable.

Alternatively, the detectable element can also be developed in

I * · * ^! tion as a complex with an intermediate chelating agent, for example, a chelating agent that forms a chelate complex that makes the element soluble in the physiological pH of the BINDING, but is less stable in having thermodynamic hands than CHELATE. An example of this intermediate chelating agent is 4,5 "hydroxy-1,3-benzene-disulfonic acid (Tiron).

In such a process, the detectable element switches the ligand.

The CHELATES OF THE INVENTION can also be produced by binding to each other the chelating agent complexed with the detectable element, and a somatostatin peptide in protected or unprotected form and, if desired, eliminating at least one protecting group that is present. The same reaction can be carried out with a detectable metal ion and then in the resulting complexed peptide the metal ion can be replaced with the desired detectable element.

The CHELATES OF THE INVENTION can also be produced by jointly linking a chelating agent complexed with a detectable element, and a somatostatin peptide fragment comprising at least one amino acid in protected or unprotected form, and then continuing the peptide synthesis step by step, until obtaining the final peptide sequence and, if desired, eliminating at least one protecting group that is present. Instead of the detectable element, the complexing agent can be complexed with. an undetectable metal and this metal can be

ι ·. . · '• cS

The resultant is then replaced by a detectable element in the somatostatin peptide complex.

i í

'When the chelating group is linked via a bridge group or spacing group to the somatostatin peptide, for example, via a radical of formula (c (l), as already defined above, both the peptide; somatostatin, as the peptide fragment or the binding agent may [comprise said bridging or spacer group.

The above-mentioned reactions can be carried out in a manner analogous to known methods. The effectiveness of the marking, depending on the chelating group present, can approach 100%, so that purification is not necessary. Radio isotopes, such as Tcnécio-99m, can be used in their oxidized form, for example, Tc-99® pertechnetate, which can be complexed under conditions of reduction.

The above reactions are conveniently carried out under conditions which avoid contamination of _the metal traces preferably are used denionizada distilled water, ultrapure reagents, chelating gradually radioactivity, etc. to reduce the effects of trace metals. CHELATES OF THE INVENTION can exist in free form or in salts. Salts include acid addition salts, such as organic acids, polymeric acids or inorganic acids, for example, hydrochlorides and acetates and forms of salts that can be obtained with the groups of carboxylic acids present in the molecule, which do not participate in the formation of the chelator, for example alkali metal salts [such as potassium or sodium, or substituted or unsubstituted ammonium salts.

The chelates of the invention and the pharmaceutically acceptable salts require pharmaceutical activity and are therefore useful both as imaging agents, for example, visualization of positive somatostatin receptor tumors and metastases, when complexed with a metal emitting ion. of paramagnetic gamma radiation or a positron emitting radio isotope or a pharmaceutical radio isotope for the in vivo treatment of positive somatosus receptor tumors23

Τ 'Λ?

tatin, when complexed with alpha or beta radioisotopes, as indicated by the standard assays.

In particular, the CHELATES OF THE INVENTION have an affinity for the somatostatin receptors, expressed or overexpressed by tumors and metastases, as indicated by the standard in vitro binding assays.

ί

A positive tumor of somatostatin receptor originated from the human gastrointestinal tract is removed from a patient and immediately placed on ice and, in a maximum time of 30 minutes, is frozen at -80 ° C. To subsequently autoradiograph, this frozen material is cut in a cryostat (Leitz 1720) in sections of 10 microns, mounted on microscopic slides previously cleaned and stored at -20 ° C for at least 3 days to improve the adhesion of the tissue to the slide. . The sections are; pre-incubated in a Tris-HCl buffer (5θ mM, pH 7.4) comprising CaCl I (2 mM) and KC1 (5 mK) for 10 minutes at room temperature and are then washed twice for 2 minutes, in the same buffer, without adding additional salts. The sections are then incubated with a CHELATO j OF THE INVENTION for two hours, at room temperature, in a buffer | Tris-HCl (170 mM, pH 7.4) which comprised bovine serum albumin (10 g / l), bacitracin (40 mg / l) and MgClg (. 5 mM) for inhibiting endogenous proteases. No specific binding was determined by adding the unmarked, unmodified somatostatin peptide to a concentration of 1. The incubated sections are washed twice for minutes, in a cold incubation buffer that contained 0.25 g / l BSA. After a brief infusion in distilled water to remove salts, the sections are quickly dried and exposed to / H H / -LKB films. After an exposure time of one week on X-ray cassettes, it was observed that the CHELATES OF THE INVENTION, for example a radioisotope CHELATE, gave very good results in the marking of tumor tissues, without marking the surrounding healthy tissues when added to a current in the range 10 to 10 M.

The affinity of the CHELATES OF THE INVENTION for somatostatin receptors can also be revealed by in vivo tests.

·, Rats with positive somatostotin receptor tumors

Pancreatic I of transplanted exocrine are treated with an intravenous injection of a CHELATE OF THE INVENTION. The compound was injected into the penis vein. Immediately after administration, the animals are healthy! placed in a gamma camera collimator and the distribution of radioactivity is monitored at various time intervals. !

The biodistribution of radioactivity can also be determined by the serial sacrifice of a number of rats so treated and the radioactivity in the organ determined.

After administering the CHELATE DA, INVENTION, for example a radioisotope CHELATE, for example gamma radiation emitter, at a dosage between 1 to 5 / * g / kg of the BINDING with an amount between 0.1 to 2 mCi of the radioisotope, the tumor becomes detectable together with organs where the excretion takes place essentially.

Consequently, in a series of specific or alternative realizations.3, the present invention also develops:

1. A process for the in vivo detection of positive tumors or somatostatin receptor metastases in an individual, comprising

a) administering a CHELATE OF THE INVENTION to said individual, and

b) the registration of the location of the receivers marked by said CHELATE.

The CHELATES of the invention for use in the in vivo detection process of the invention are the CHELATES complexed with radio isotopes emitting gamma radiation, positrons or paramagnetic metal ions, for example, as already mentioned above.

The CHELATES OF THE INVENTION for use as imaging agents in the process (1) can be administered parenterally, from;

preferred intravenously, for example in the form of injectable solutions or suspensions, preferably in a single dose. The appropriate dosage will of course vary depending, for example, on the LIGANDO and Upo of the detectable element used, for example, the radio isotope. The appropriate dose to be injected is in the range that provides the image by means of known techniques of photo-research. When a radio-labeled CHELATE OF THE INVENTION is used, it can advantageously be administered in a dose that has radioactivity in the range of 0.1 to 50 mCi, preferably in the range of 0.1 to 30 mCi, and preferably between 0.1 at 20 mCi. A dosage range indics.da can be from 1 to 200 µg of the LIGANDO marked with 0.1 to 50 mCi, preferably between 0.1 to 30 mCi, for example 3 to 15 mCi, radioisotope emitting radiation- gamma, depending on the radioisotope-gamma used. For example with In, the use of radioactivity in the lower range is preferred, whereas with Tc the use of radioactivity in the upper range is preferred.

enrichment of tumor areas with CHELATES can be followed by corresponding imaging techniques, for example using medium nuclear imaging instrumentation, for example a screen, a gamma camera, a gamma rotating camera, each of which is preferably assisted by a computer; PET screen (proton emission tomography); HRI equipment or TAC equipment.

The CHELATES OF THE INVENTION, for example most of the gamma-emitting CHELATES are substantially eliminated through the kidneys and do not accumulate significantly in the liver.

2. A process for in vivo treatment of somatostatin receptor positive tumors and tetastases in an individual in need of such treatment, comprising administering to said individual a therapeutically effective amount of a CHELATE OF THE INVENTION.

The CHELATES OF THE INVENTION, for use in the in vivo treatment process of the invention, are chelates complexed with alpha or beta radioisotopes as defined above.

/] f »-a

2b oresenxe wJ The dosages used in the practice of the therapeutic process of winning will, of course, vary depending on, for example, the particular condition to be treated, for example the tumor volume, the particular QUSLATO used, for example the average QUSLATO lifetime in the you— immor, and the desired therapy. In general, the dose is calculated based on the distribution of radioactivity for each organ and the increase in the observed signal. For example, QUSLATO can be administered in a daily dose range J, which has a radioactivity of about 0.1 to 3 mCi / Kg of body weight, for example 1 to 3 mCi, preferably between 1 to 1.5 mCi / kg of body weight. An indicated daily dosage range is between 1 to 200 of the LIGANDO marked with 0.1 to 3 mCi / kg of body weight, for example, from 0.1 to 1.5 mCi / Kg of body weight of the radioisotope alpha or beta, conveniently administered in dojses divided up to 4 times a day. !

I

The QUBLATES OF THE INVENTION emitters of alpha or beta radiation can already be administered by any conventional route, in particular parenterally, for example in the form of injectable solutions or suspensions. They can also be administered by infusion, for example an infusion lasting 30 to 60 minutes. Depending on the location of the tumor, they can be administered as close as possible to the tumor area, for example by means of catheters. The mode of administration chosen may depend (I on the dissociation rate of the CHELATE used and the rate of excretion.

The QUSLATES OF THE INVENTION can be administered in free form or in a pharmaceutically acceptable form. Such ss.is can be prepared in a conventional manner and exhibit the same level of activity as the free compounds. '| The CHELATES OF THE INVENTION for use in the process of the present invention can preferably be prepared shortly before administration to the subject, i.e., radiolabeling with the desired detectable metal ion, particularly the alpha beta or gamma isotope, can be performed shortly before administration.

The CHELATES OF THE INVENTION can be adapted to provide images i

/) or treat tumors such as pituitary, gastro-entero-pancreatic, central nervous system, breast, prostate, ovarian or colonic, small cell lung cancer, paragangliomas, neuroblastomas, pheochromocytomas, medullary thyroid carcinomas, myelcmas, etc. ., and its metastases. !

According to other embodiments of the invention, the CHELATES OF THE INVENTION, emitters of gamma radiation, can also be used as image-prohibiting agents for assessing renal function.

Groups of 5 rats were used. Each rat was injected intravenously, into the tail vein, with 0.1 ml comprising 1 mCi of 1 CHELATE DA! INVENTION. The rats are placed in metabolic cages to collect urine. After 10 or 120 minutes of the injection, the ureters are ligated and the rats are anesthetized with chloroform. The image of the uropoietic system is performed using the usual imaging technique. In this test, the CHELATES OF THE INVENTION, emitters of gamma radiation, improve the image of renal excretion when administered at a dosage ranging from 0.1 to 30 mCi.

Accordingly, the present invention further develops a process for the in vivo assessment of an individual's renal function, which comprises administering to that individual an effective amount of a gamma radiation emitting CHELATE and recording renal function.

According to other aspects3 of the invention, it was developed:

i. A pharmaceutical composition comprising a BINDING OF THE INVENTION, in the free form or in a pharmaceutically acceptable salt, together with a

I or more pharmaceutically acceptable carrier substances or thinners; ii. A pharmaceutical composition comprising a QUE-;

I

LATO according to the invention, in free form or pharmaceutically acceptable salt, together

2tí / · / í

one or more diluents or carrier substances; acceptable.

Such compositions can be produced in a conventional manner.

A composition according to the invention can also be presented in a separate package, with instructions for mixing the ligand with the metal ion and for administration of the resulting CHELANT. It can also be presented in the form of a double pack, that is, a single pack containing separate unit doses of the LIGANDO and the detectable metal ion, with instructions for mixing them and for the administration of the CHELATE.

Diluents or carrier substances may be present in unit dosage form.

In the following examples, all temperatures are shown in ° C and the values of / <7 ^{2 <} ^ D - have not been corrected. The following abbreviations are already used:

Boc terc, -butylcabonyl

TFA trifluoroacetic acid

DTPA diethylenetriamine-penta-acetic acid

EXAMPLE 1: DTPA-DPhe-Cys-Phe-Dtrp-Lys-Thr-Cys-Thr-ol

1.1 g of Dphe-Cys-Phe-Dtrp-Lys (-Boc) -Thr-Cys-Thr-ol in the free base (1 mM) were dissolved in 5 l of dioxane / H 2 O 1: 1 (v / v) and reacted with 5 g of NahCO3. 520 mg of di-anhydrous DTPA was added slowly with stirring. The reaction mixture was stirred for an additional 30 minutes and freeze-dried. The residue was dissolved in 250 ml of water and the pH adjusted to 2.5 concentrated HCl. The precipitated product was removed by filtration, washed and dried over phosphorous pentoxide. After chromatography on a column of silica gel, the following are isolated

I -—— | I products: 230 mg DTPA-DPhe-Cys-Phe-DTrp-Lys-Tbr-Cys-Thr-ol and 5®6rrg

Ζ)

Λ / of the corresponding dimer DTPA- (DPhe-Cys-phe-Dtrp-LysÇ-Boc) -thr-Cys-ol) 2.

i -—- 3 ml of TFA was mixed with 200 mg of DTPA-DPhe-Cys-Phe-Dtrp-Lys - (- Boc) -Thr-C ^ s-Thr-ol. After 5 minutes at room temperature, the mixture) was precipitated with diisopropyl ether, removed by filtration and dried. The residue was desalinated on Duolite and lyophilized, to obtain 150 mg of the title compound:

/ c (J ²⁰ D = 26.6 ° (c = 1.95% AcOH)!

starter material can be produced as follows:!

a) H-Dphe-C ^ s-Phe-Dtr £ -Ly £ - £ .b £ cj_-thr-çj £ -thr- £ l

Slowly, 25% di-tert-butyl-pyrocarbonate, dissolved in 30 ml of DMF, are added dropwise at room temperature to a solution ~ 1 -—- ¹ ->

10 g of H-Dphe-Cys-Fhe-Dtrp-Lya-Thr-Qys ^ fchr-olem acetate 100 ml DMF. Two hours later at room temperature, the solvent is removed in vacuo, washed with diisopropyl ether and dried. 0 crude product is purified by chromatographic '' silica gel (eluent: CH ₂ Cl / MeOH 9: 1) and then isolated as an amorphous white ρδ.

A7 ²⁰ - 29.8 ° C (c ₌ 1.28 in DMF).

D

EXAMPLE 2: DTPA- (DPhe-C.ys-Phe-DTrp-Lys-Thr-gys-Thr-cl)

The fraction containing the intermediate product DTPA-ÇDPhe-Cys-Phe-Dtrp-1

-LysC-Boc -) - Thr-Cys-Thr-cl), obtained in Example 1, was treated as described above, to obtain the corresponding monomeric form, the Boc protecting groups were removed, to contain the title compound :

= -24.5 ° C (c - 0.55 95 °] the AcOH). j

EXAMPLE 3? H ₂ M CH ₂ ) -5-CO-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-ol

The. 0.56 g of H-BPhe-Cys-Phe-DTrp-Lys (BOC) -thr-Cys-Thr-ol, 0.5 mmol of Fmoc-c.-amir.ocapoíco acid - and 115 mg of hydroxy-benzotriazole were: dissolved in 10 ml of DMF and cooled to -30 ° G. To this solution, a solution of 115 ml of dichlorohexylcarbodiimide in 5 ml of DMF (cooled to -10 ° C) was added.

After the reaction time of 24 hours, during which i j

the temperature reached room temperature, the ôiciclohexilurea! The resultant was removed by filtration and the filtrate was diluted with water to 10 x its volume. The precipitated reaction product was removed by filtration, washed and dried over phosphorus pentoxide. The crude product was used without further purification for the next faBe.

B. Ebi the c-cleavage

0.5 g of the crude product obtained in the ligation reaction (a) were treated for 10 minutes at room temperature with 5 ml of DM4 / piperidine 4: 1 v / v (clear solution) and subsequently mixed with 100 ml of di -isopropylether. The reaction product, which then precipitated, was removed by filtration, washed and dried.

This crude product was used without further purification in the next stage. )

ç. BOC-cleavage!

300 g of the crude product obtained in (l.b) were treated for 30 minutes

at room temperature, with 5 ml of 100% THF (completely dissolved) and then mixed with 50 ml of diisopropyl ether. After adding 2 ml of HCl / diethyl ether, the resulting deposit was renewed by filtration, washed and dried under high vacuum.

final product was purified by chromatography on silica gel (CHC13 / / ΜβΟΗ / ϊΚΟ / ΑοΟΗ 7: 3: 0.5: 0.5), with subsequent desalination on Duoli31

te (gradient: ^ O / AcOH 95 ^ 5) --- H ^ O / dioxane / AcOH 45 * 50 * 5).

The title compound was obtained as an acetate (white lyophilisate).

7 rt 7 ² θ ^L = -32 ° C (c = O ,; 555I AcOH).

The resulting compound can be used for the reaction with DTPA according to the technique of Examples 1 and 2.

EXAMPLE 4:

CONNECTING the following can be prepared following the techniques sold in Examples 1 and 3:

i π

DTPA- ^ Ala-DPhe-Cys-Pbe-DTrp-Lys-Thr-Gys-Thr-ol.

_y 20

L - - 14.8 ° (c ₌ 0.5 95 »AcOH).

I - - j |

EXAMPLE 5s In DTPA-DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Tbr-ol DTPA-DPhe-Cys-Pbe -DTrp-Lys-1hr-Cy's-Thr-ol is dissolved in 5 ml 0.01 M acetic acid _o The resulting solution is filtered through a 0.22 µm filter. Millex-GV, divided into portions of 0.1 ml and ar-; stored at -20 ° C. 0 ^^^ InC13 (Amersham, 1 mCi / 100 µl) is pre-diluted in an equal volume of 0.5 M sodium acetate and the marking is carried out by mixing the LIGANDO with the InCle solution by smoothly homogenizing it,

at room temperature ₀

An HSPES buffer, pH 7.4, was then added to make the solution 10 ^_6 M.

, ί

Λ

EXAMPLE 6: labeled DTPA-Dphe-Gys — Phe-DTrp-Lys-Thr-G.ys-Thr-ol ο 9¾ is obtained from the radio isotope generator ^ θδΓ- ^ θΥ. !

!

The construction of the generator, its elution and the conversion of the / ^ ° T ^ EDTA into the acetate complex was carried out according to the process revealed by

M. Chinol and D. J. Hnatowich, in J. Nucl. Med. 28, 1465-1470 (1987). 1 mg of 0.01 µ acetic acid, allowed to warm to room temperature and 1.0 mCi of Y in 50 µl of sterile 0.5 M acetate was added. The mixture was then left to stand for an interval of time from 30 minutes to 1 hour, to maximize chelation.

One of the groups of INVEKÇSO LIGANDS are the somatostatin peptides, for example the somatostatin analogs, which have at least one of the amino acid units of the chelating group that is linked to said amino group by an amide bond, in the free form or salt.

One of the groups of the CHELATES of the INVENTION are the LIGAND03 already mentioned above, complexed with a detectable element, for example a metal ion, in the free form or in the form of a salt.

Claims (3)

1 * - Process for the preparation of a somastatin peptide, having at least one chelating group, for a detectable element, the said group being linked to an amino group of said peptide, either directly or indirectly and without having the aforementioned amino group no significant binding affinity for soma-receptors; tostatin or in the form of salt, characterized by the fact that it comprises the operations consisting of a) if at least one protecting group that is present in a somastotatin peptide has a chelating group or! h) if they link to each other, by means of an amide bond, two fragments of peptide that each contain at least one amino acid or an amino alcohol, in protected form and one of them has the chelating group and in which the amide bonding is carried out in order to maintain the desired amino acid sequence, and then optionally carry out operation a) of the process or c) if a chelating agent and the desired somatostatin peptide are attached to each other, in protected form or unprotected so that the chelating group is fixed on the desired N-amino group of the peptide, and then optionally a) j or d) if a functional group is removed from a protected or unprotected peptide, which has a chelating group, or the group is transformed into another functional group, so that another protected γίο or unprotected peptide is obtained, which has a chelating group and, in the latter case, if procedural operation a) is carried out; or e) oxidizing a modified somatostatin peptide with a chelating group in which the mercapto groups of the Cys radicals are free, to produce an analog in which the two Cys radicals are linked by an SS bridge, and recover the compound thus obtained in the free form or in the form of salt. 28. The method of claim 1 for preparing a somatostatin peptide, in which the chelating group is attached to the peptide: indirectly by a spacer group, preferably a radical i of the formula in which · represents alkylene in alkenylene in ^ “^ Ll ^or j where R 'is the residue attached at the alpha position to a natural or synthetic alpha-amino acid; Z is a functional group capable of reacting covalently with a chelating group, characterized by the fact that it comprises the following operations | reacting a peptide or a peptide fragment, free of a bridging group or a spacing group, with a respective compound formed of a bridging group or a spacing group; if a chelating agent and said modified peptide or a peptide fragment are bound together, in protected or unprotected form, so that the chelating group is fixed in the desired N-amino group and, if necessary, eliminated by least one protecting group, which is present in said peptide <> 3. A process according to claim 2, characterized in that it comprises the reaction of the chelating agent with a bridging group or spacing group forming agent, before the modified chelating agent is bound to each other. and the desired somatostatin peptide in protected or unprotected form. 4 - Process according to any preceding claim wherein the somatostatin peptide is already a compound of formula I Λ r \ ’ A 'C ^ -S ^ Y ₂ -S-CH ₂ .NC HC OBC -DS-IIH-G HG in which A represents C ^ -C ^ ₂ alkyl, β7 ~ ^ ΐθ ^{033 13111} S ^ru P ° <ie RCO- formula, in which i) D hydrogen, alkyl in, phenyl or phenylalkyl in Ογ-Ο ^ θ, or ii) RCO- is a) a L-phenylalanine or D-phenylalanine residue optionally substituted on the ring by F, Cl, Br, NO ₂ , NH ₂ , OH, C 4 -C 4 alkyl and / or C 4 -C 4 alkoxy h) the residue of an alpha — natural or synthetic amino acid, different from that mentioned in a), or of a corresponding D — amino acid, or c) a dipeptide residue, in which the individual residues of the amino acid are the same or different and are chosen from those already defined above in) and / or in h) j optionally, the alpha-amino group of the amino acid residues a) and h) and the N-terminal amino group of the dipeptide residues c) are monoalkylated or dialkylated by C ^-C ^ ₂ alkyl or substituted by C ^--CCg alkanyl THE.' is hydrogen, C ^-C ^ _{2 2} ° alkyl or phenylalkyl in Y ^ and Y ₂ together represent a direct bond or each of the symbols Y ^ or Y ₂ is independently hydrogen or a radical of the formulas (l)