HK1155363A - Novel dual targeting antitumoural conjugates - Google Patents

Description

Novel dual targeting antitumor conjugates

Technical Field

The invention relates to dual targeting cytotoxic derivatives and their preparation. The compounds described have a tumor-specific action and incorporate 3 functional units: a tumor recognition moiety and a tumor-selective enzyme substrate sequence. These conjugates are designed to ensure serum stability and at the same time the desired effect in tumor cells due to enzymatic cleavage.

Prior Art

Traditional cancer chemotherapy is based on the assumption that rapidly proliferating cancer cells are more likely to be killed than quiescent cells of physiological tissues. Indeed, cytotoxic agents have very poor specificity, which causes serious adverse effects. Over the past 30 years, various systems have been explored to selectively deliver drugs at their site of action. Recent advances in the understanding of the typical receptors that are overexpressed during cancer cell proliferation have allowed the development of selective ligands that, when conjugated to a cytotoxic agent, preferentially and differentially direct the cytotoxic agent to the tumor. Unlike typical prodrugs, the linker between the ligand and the drug must be stable in circulation and should be readily cleavable by chemical or enzymatic mechanisms to regenerate the cytotoxic agent after internalization of the entire conjugate into cancer cells.

Recent advances in tumor-targeted drug conjugates have required monoclonal antibodies, polyunsaturated fatty acids, hyaluronic acid, and oligopeptides as ligands for tumor-associated receptors.

Today, several immunoconjugates (immunoconjugates) are undergoing clinical trials: maytansine (Maytansin; Liu c., et al., proc.natl.acad.sci., 1996, 93, 8618), doxorubicin (doxorubicin; Saleh m.n., et al., j.clin.oncol., 2000, 18,112282), herceptin (herceptin; baselga j, et al, j.clin.oncol., 1996, 14, 737), calicheamicin (calicheamicin; bross p.f., et al, clin.cancer res, 2001, 7, 1490; chan s.y., et al., Cancer immunol.immunoher., 2003, 52, 243). With respect to the latter, Mylotarg (i.e., calicheamicin linked to CD33 antibody) has been approved by the U.S. Food and Drug Administration (FDA) in the year of the official 2000 for marketing for the treatment of acute leukemia (Hammann p.r., et al, Bioconjugate chem., 2002, 13,1，47)。

practical use of immunoconjugates is only suitable for highly effective drugs, since a limited amount of antigen is overexpressed on the surface of tumor cells, and each monoclonal antibody (mAb) can only be loaded with a limited number of molecules without reducing binding affinity and increasing immunogenicity.

Recently, many conjugates of cytotoxic agents with oligopeptides that target different receptors overexpressed by tumor cells have been investigated as possible selective anti-tumor chemotherapeutic agents. Among the many oligopeptides that appear to be most promising are somatostatin (somatotatin; Pollak m.n., et al, proc.soc.exp.biol.med., 1998, 217, 143; fuseier j.a., et al, bioorg.med.chem.lett., 2003, 13, 799), bombesin (bombein; Moody t.w., et al, j.biol.chem., 2004, 279, 23580), integrin (integrin) -mediated RGD peptides (WO200117563, ruoslahte, Nature reviews, Cancer, 2002, 2, 83; Dickerson e.b., et al, mol.cancer res, 2004, 2,12663; de Groot f.m., et al, mol.cancer ther, 2002, 1, 901; chen x., et al., j.med.chem., 2005, 48, 1098). Chemical linkers commonly tested between tumor recognition moieties and anticancer drugs include hydrazones, disulfides, and peptide substrates of lysosomal enzymes (peptides).

The nature of the linker is a prerequisite to determine the in vivo fate, stability, solubility and bioavailability of the conjugate.

The tumor targeting conjugates of the present invention are made of 3 functional units (tumor recognition moiety and anticancer drug) linked together by a spacer (linker).

WO05111064 filed in the name of the applicant describes cyclic peptides exhibiting an RGD unit and possessing anti-integrin activity. WO05111063, filed in the name of the applicant, reports 7-iminocamptothecin derivatives conjugated via a spacer to cyclopeptides recognizing integrins.

WO05110487, filed in the name of the applicant, reports camptothecin derivatives conjugated at position 20 to integrin antagonists.

Disclosure of Invention

It is an object of the present invention to develop a pharmaceutical composition comprising integrin alpha linked to a cytotoxic drug via a novel molecular bridge_vβ₃And alpha_vβ₅A tumor targeting conjugate that recognizes a moiety, the molecular bridge comprising 3 units. The latter (i.e., the molecular bridge) is composed of a spacer, a peptide cleavable by a tumor-associated enzyme, and a self-immolative functional unit.

The spacer chosen is made of a small flexible ethylene glycol surrogate containing a hydrophilic amino acid or heterocyclic structure that functions as a rigid moiety that imparts solubility to the entire conjugate and does not interfere with binding to the receptor. These specific spacers are superior to the widely used high molecular weight glycols which, although having excellent solubility properties, are not suitable because of the tendency to form rings which interfere with the binding domains.

A number of linker-containing peptides have been disclosed as substrates for cathepsin B, e.g., Phe-Lys, Val-Cit (Dubowwick G.M., et al, Bioconjugate chem., 2002, 13,4，855)；Gly-Phe-Leu-Gly(Rejmanova P.，et al，Biomaterials，1985，6，145); D-Ala-Phe-Lys (de Groot f.m.h., et al, mol.cancer.ther., 2002, 1, 901). Certain of these peptides have been successfully used in conjunction with antibodies which, on their own, protect them from the action of plasma peptidases due to their bulky nature. However, when we tested these peptide sequences (e.g., oligopeptides) for conjugates containing small ligands, it was found that these peptide sequences were immediately cleaved to release the cytotoxic agent into the circulation, contrary to what was described by other authors. In particular, the peptide containing the Phe-Lys linker (ST3280) showed a high degree of instability in the various assays performed. Dubowchip document cited in the previous disclosureA cathepsin B labile dipeptide ligand. These same authors also showed another correlation 4 years ago when Cit amino acids were in P₁At position of P₂The study of the influence of amino acids at a position, concluded that the optimal amino acid at this position is Val (dubowchip g.m., et al, bioorg.med.chem., 1998, 8, 3341) due to hydrophobic interactions in the binding site of cathepsin B, while analogues containing Val instead of Ala contribute to a significant slowing down of doxorubicin release, a result which is clearly contrary to the objective of the study.

Surprisingly, it has now been found that: Ala-Cit or D-Ala-Cit, which surprisingly appears to be stable in murine blood and cleavable in tumor cells, is particularly suitable as a means for releasing cytotoxic motif motifs (motif) at the site of action.

The presence of self-immolative groups is also necessary to enhance the action of endopeptidases (Carl p.l., et al, j.med.chem., 1981, 24,5，479；Shamis M.L.，et al.，J.Am.Chem.Soc.，2004，126，6,1726). These novel linkers better guarantee the desired pharmacological properties of the related conjugates, such as metabolic stability and further release of cytotoxic agents upon internalization in cells, as well as optimal solubility and bioavailability. Furthermore, these linkers have been designed to have a size and configuration suitable for binding of the targeting device to the receptor.

These novel linkers are labile molecular bridges that can be applied to a variety of different ligands and different antineoplastic agents.

The invention comprises compounds of the general formula I

[(L-D)_nE]_m-F-D-PI-SI-CT

Formula I

Wherein

L is a cyclic peptide of formula II which recognizes the alpha-integrin receptor

c(R¹-Arg-Gly-Asp-R²)

Formula II

R¹Is Amp, Lys or Aad;

R²is Phe, Tyr or Amp in the R configuration;

d may be the same or different at each occurrence and is absent or a divalent radical of formula III

-SP¹-A¹-SP²-A²-SP³-

Formula III

SP¹Is absent or R³-(CH₂)_q-(OCH₂-CH₂)_q-O-(CH₂)_q-R⁴；

R³And R⁴Are identical or different and are absent or are a divalent radical of the formula IV, VIII or IX

q may be the same or different at each occurrence and is independently an integer from 0 to 6;

A¹is a natural or non-natural (L) or (D) amino acid, absent or containing a hydrophilic side chain;

SP²is absent or associated with SP¹The same;

A²is absent or is in combination with A¹The same;

SP³is absent or associated with SP¹The same;

m is 1 or 2;

n is 1 or 2;

e may be the same or different at each occurrence and is Glu, Lys or absent;

f is the same as or absent from E or is a histidine analog of formula X;

wherein the triazole ring is attached to a D-PI-SI-CT moiety, the carbonyl moiety is attached to a L-containing moiety and SP¹Is as defined above;

PI is a natural or non-natural oligopeptide consisting of (L) or (D) amino acids selected from Ala or Cit;

SI is a divalent radical p-aminobenzyloxycarbonyl;

CT represents a cytotoxic group;

their tautomers, geometric isomers, optically active forms (such as enantiomers, diastereomers and racemic forms thereof) and pharmaceutically acceptable salts thereof;

the premise is that:

at least one D should be present;

and E is linked via its amino moiety to the moiety containing the L group when E is present and Lys, or via its carboxy moiety to the moiety containing the L group when E is present and Glu.

One embodiment of the invention is a compound of formula I, wherein CT represents a camptothecin derivative.

Another embodiment of the invention are compounds of formula I, wherein CT represents a camptothecin derivativeSubstance, R¹Is Amp and R²Is Phe.

Another embodiment of the invention are compounds of formula I, wherein PI represents an oligopeptide comprising 2 or 3 amino acid residues.

Another embodiment of the invention are compounds of formula I wherein m ═ 1 and n ═ 1.

Another preferred embodiment of the invention are compounds of formula I, wherein m ═ 1 and n ═ 2.

The compounds of formula I can be prepared using standard coupling procedures known to those skilled in the art. It is to be understood that unless otherwise indicated, it is appropriate to give typical or preferred experimental conditions (i.e., reaction temperature, time, moles of reactants, solvent, etc.), and that other experimental conditions may be used unless otherwise indicated. The optimum reaction conditions may vary depending on the particular reactants or solvents used, but these conditions can be determined by one skilled in the art by routine optimization procedures.

The invention further provides a process for preparing compounds of the general formula I, for example by reacting the free amino group of the PI fragment of a compound of the formula V

(CT-SI-PI)-NH₂(formula V)

(wherein CT, SI and PI are as described above)

With azide-containing derivatives of formula VI

L-(SP¹-A¹-SP²-A²-SP³)-N₃(formula VI)

(wherein L, SP¹、A¹、SP²、A²And SP³Is as described above and R⁴Is CO) reaction.

Alternatively, compounds of formula I may be prepared by reacting compounds of formula VII, as described in the document Rostovtsev v.v., et al, angelw.chem., 2002, 41, 2596

(CT-SI-PI) -CO-C ≡ CH (formula VII)

(wherein CT, SI and PI are as described above)

With a compound of formula VI (wherein L, SP is present in the compound of formula VI)¹、A¹、SP²、A²And SP³Is as described above, provided that R⁴Is absent) reaction.

Also by reacting a compound of formula XI

(CT-SI-PI)-D-NHCH₂-C ≡ CH (formula XI)

(wherein CT, SI, PI and D are as described above)

With compounds of the formula XII

[(L-D)_nE]_m-COCH₂-N₃(formula XII)

(wherein L, D and E are as described above) to produce a compound of formula I.

Alternatively, by reacting a compound of formula XIII

(CT-SI-PI)-D-N₃(formula XIII)

(wherein CT, SI, PI and D are as described above)

With compounds of the formula XIV

[(L-D)_nE]_m-CO-CH(NHD)CH₂-C ≡ CH (formula XIV)

(wherein L, D and E are as described above) to produce a compound of formula I.

The amino acid having a hydrophilic side chain means an amino acid selected from arginine, asparagine, aspartic acid, citrulline, cysteine, glutamic acid, glutamine, histidine, lysine, serine, threonine or tyrosine.

Camptothecin derivatives or cytotoxic groups refer to camptothecin, such as the derivatives described in WO00/53607 and WO04/083214, filed in the name of the applicant.

Another object of the present invention is a method of treating a mammal suffering from an uncontrolled cell growth, invasion and/or metastasis condition comprising administering a therapeutically effective amount of a compound of formula (I) as described above. The term "therapeutically effective amount" as used herein refers to the amount of therapeutic agent required to treat or alleviate the subject disease or indication or to manifest a detectable therapeutic effect.

For either compound, the therapeutically effective dose can be estimated initially by cell culture assays or animal (typically mouse, rat, guinea pig, rabbit, dog or pig) models. The animal model may also be used to determine appropriate concentration ranges and routes of administration. This information can then be used to determine effective dosages and routes of administration for use in humans. In calculating the Human Equivalent Dose (HED), it is recommended to use the conversion tables provided in the literature guidelines for Industry and reviews (2002, U.S. food and Drug Administration, Rockville, Maryland, USA).

The precise effective dose for human administration will depend on the severity of the disease state, the general health of the individual, age, weight and sex, diet, time and frequency of administration, drug combination, response sensitivity and tolerance/response to therapy, etc. Such dosages may be determined by routine experimentation and at the discretion of the clinician. Generally, an effective dose will be between 0.01 and 100mg/kg (preferably 0.05 to 50 mg/kg). The compositions may be administered to the patient individually or may be administered with other agents, drugs or hormones.

For administration of the therapeutic agent, the medicament may also contain a pharmaceutically acceptable carrier. These vectors include antibodies and other polypeptides, genes, and other therapeutic agents (such as liposomes), provided that the vectors themselves do not induce the production of antibodies harmful to the individual receiving the composition and can be administered without undue toxicity.

Suitable carriers may be slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polyamino acids, amino acid copolymers and inactivated virions.

A complete discussion of pharmaceutically acceptable carriers is found in Remington's Pharmaceutical Sciences (Mack pub. Co., N.J.1991).

The pharmaceutically acceptable carrier in the therapeutic composition may additionally contain liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may be present in these compositions. For ingestion by a patient, such carriers enable such pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries (suspensions), suspensions and the like.

Once formulated, the compositions of the present invention can be administered directly into the body of an individual. The subject to be treated may be an animal; in particular, it can be used for treating human body.

The agents of the present invention may be administered by a number of routes including, but not limited to, oral, intravenous, intramuscular, intraarterial, intraspinal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal, rectal routes or topical application to diseased tissue following surgery.

The dose treatment may be a single dose course or a multiple dose course.

Another object of the present invention is a pharmaceutical composition containing as active ingredient at least one compound of formula (I) in an amount such as to produce a significant therapeutic effect. The compositions encompassed by the present invention are entirely conventional and are prepared using methods commonly practiced in the pharmaceutical industry. These compositions are in solid or liquid form and are suitable for oral, parenteral or intravenous administration, depending on the route of administration adopted. The compositions of the invention comprise the active ingredient and at least one pharmaceutically acceptable carrier or excipient.

Brief description of the drawings

FIG. 1 depicts the chemical structures of different fragments used to synthesize the dual targeting cytotoxic derivative class.

FIG. 2 depicts the chemical structures of dual targeting cytotoxic derivative species.

FIG. 3 depicts the synthesis of certain building blocks for the synthesis of fragments 1, 2, 5, 6 and 12 and the fully synthesized fragment 10 (FIG. 3. e).

Figure 4 schematically depicts the properties of the two fragments required for the synthesis of each final compound.

The following illustrative examples are in no way intended to be exhaustive of the invention.

Examples

For short:

aad: aminoadipic acid

And (3) Alloc: allyloxycarbonyl radical

Amp: p-amino methyl phenyl aniline

Boc: tert-butyloxycarbonyl radical

Cit: citrulline

CPT: camptothecin

DCM: methylene dichloride

DIPEA: diisopropylethylamine

DMF: dimethyl formamide

equiv.: equivalent weight

Et₂O: ether (A)

Fmoc: 9H-fluorenylmethoxycarbonyl

HCTU: (2- (6-chloro-1H-benzotriazol-1-yl) -1, 1, 3, 3-tetramethylammonium hexafluorophosphate)

HOAt: 1-hydroxy-7-azabenzotriazoles

HOBt: 1-hydroxybenzotriazoles

MALDI: matrix-assisted laser desorption ionization

MeOH: methanol

NMP: n-methyl pyrrolidone

PABA: 4-aminobenzyl alcohol

PABC (polyamide resin): para aminobenzyloxycarbonyl

Pmc: 2, 2, 5, 7, 8-pentamethyl-chroman-6-sulfonyl

RP-HPLC: reversed phase high performance liquid chromatography

RT: at room temperature

rt: retention time

SPPS: solid phase peptide synthesis

TBTU: o- (benzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium tetrafluoroborate

TEA: triethylamine

TFA: trifluoroacetic acid

Tof: time of flight

General remarks:¹the H spectrum is in the indicated DMSO-D₆、CDCl₃Or D₂O solution and recorded at 300MHz on a Bruker instrument. The chemical shift values are expressed in ppm and the coupling constants are in Hz. Flash column chromatography was performed using silica gel (Merck 230-.

Example 1

Synthesis of ST3833

Fragment 2(1 eq) dissolved in DMF (2ml) was added to a solution of fragment 1 (prepared in situ, 0.32 mmol) and DIPEA (1 eq) in DMF (7 ml). The pH was adjusted to about 7.5 using DIPEA and the reaction mixture was stirred at room temperature in the dark. After 2 hours, another equivalent of fragment 1 was added, the pH was adjusted again and the reaction mixture was allowed to stir overnight.

By preparative HPLC (column, Discovery Bio Wide pore C18, Supelco, 250X21.2mm, 10 μm; mobile phase: 29% CH)₃After purification in aqueous CN + 0.1% TFA, λ 220nM) and freeze drying, ST3833(365mg, 97.6% purity) was obtained. The yield was 60%.

Analytical grade HPLC (Gemini, Phenomenex, C18, 250X4.6mm, 5 μm; mobile phase: 34% CH)₃Aqueous CN + 0.1% TFA, λ 220 nm). The conjugate showed two peaks at retention times of 7.96 and 10.43 minutes, which are based on a mixture of E/Z isomers of the original cytotoxic molecule. Mass of Maldi-Tof: 1650.71[ M + H]⁺。

¹H-NMR(DMSO-D₆) Main displacement, δ: 9.28,8.57,8.28,8.22,8.14,8.07,7.93,7.88,7.75,7.65,7.55,7.45,7.36,7.24,7.15,7.11,7.03,7.02,6.42,5.95,5.42,4.94,4.60,4.41,4.28,4.09,3.95,3.89,3.57,3.48,3.18,3.00-2.31,1.91,1.75,1.60-1.30,1.25,0.90.

Example 2 (for comparison)

Synthesis of ST3280

The coupling reaction of fragment 1 with fragment 3 was carried out following the procedure described in example 1 before the alloc protecting group was removed.

Bu is put into₃SnH (0.172 mmol), AcOH (0.375 mmol) and Pd (PPh)₃)₄(0.003 mmol) was added to [ Alloc-ST3280](0.078 mmol) in DMF (3 ml). The reaction mixture was stirred at room temperature under argon for 1 hour. After evaporation of the solvent under reduced pressure, the residue was subjected to preparative HPLC (column, Alltima, Alltech, R)P18, 10 μm, 250x22 mm; mobile phase: 34% CH₃Aqueous CN + 0.1% TFA). After lyophilization, the conjugate was obtained (99.9% purity). The yield was 55%.

Analytical grade HPLC (Gemini, Phenomenex, C18, 250x4.6mm, 5 μm; mobile phase: 35% CH₃Aqueous CN + 0.1% TFA; λ 360 nm). Retention time of E/Z isomer: 7.24 and 9.61 minutes. ESI mass: 1696[ M + H ]]⁺。

¹H-NMR(DMSO-D₆) Main displacement, δ: 8.57,8.28,8.22,8.14,8.07-7.50,7.36,7.24,7.20-6.90,6.42,5.42,4.94,4.60,4.41,4.28,4.18-4.00,3.95,3.90,3.57,3.48,3.12-2.25,1, 91,1.55,1.38,0.90.

Example 3

Synthesis ST4167

Mixing sodium ascorbate (0.089 mmol) and CuSO₄·5H₂A solution of O (0.009 mmole) in water (500. mu.l) was added to a solution of fragment 4(0.09 mmole) and fragment 5(88mg, 0.09 mmole) in DMF (2 ml). The pH was adjusted to pH 6 by addition of NaOH and the suspension was stirred at room temperature overnight. After evaporation of the solvent under reduced pressure, the residue is subjected to preparative HPLC (column, Alltima C18, 10 μm, Alltech; mobile phase: 33% CH)₃Aqueous CN + 0.1% TFA, λ 220 nm). After lyophilization, the desired adduct was obtained (72mg, 97% purity). The yield was 44%.

Analytical grade HPLC (column, Gemini C18, 250x4.6mm, 5 μm; mobile phase: 34% CH)₃Aqueous CN + 0.1% TFA, λ 220 nm). Retention time: 7.7 and 9.9 minutes. ESI mass: 1745.7[ M + H]⁺。

¹H-NMR(DMSO-D₆+D₂O), main shift, δ: 8.90,8.44,8.33,8.18,8.03-7.84,7.8-7.69,7.45,7.39,7.2-6.94,5.48-5.30,5.19,4.89,4.69,4.6-4.24,4.20,4.13,4.02,3.89-3.52,3.5-3.37,3.24,3.10-2.62，2.40-2.30，1.93-1.25，0.85。

Example 4

Synthesis of ST4215

The coupling reaction of fragment 4 with fragment 6 was carried out following the procedure described in example 3.

The crude reaction product obtained from the cycloaddition reaction was subjected to preparative HPLC (column, Alltima, C18, 10 μm, Alltech; mobile phase: 30% CH)₃Aqueous CN + 0.1% TFA). After lyophilization, the desired adduct was obtained (52mg, 98.6% purity). The yield was 41%.

Analytical grade HPLC (Gemini, Phenomenex, C18, 250x4.6mm, 5 μm; mobile phase: 30% CH₃Aqueous CN + 0.1% TFA, λ 220 nm). Retention time: 11.23 and 15.43 minutes. ESI mass: 2106[ M + H ]]⁺。

¹H-NMR(DMSO-D₆) Main displacement, δ: 9.79,9.13,8.42,8.15,7.95,7.86,7.80-7.69,7.45-7.39,7.18-6.70,5.47-5.24,4.85,4.60-4.30,4.28-3.65,3.64-3.31,3.30-2.61,2.43-2.30,1.91-1.38,1.33,0.84.

Example 5

Synthesis of ST5548TF1

The cycloaddition reaction of fragment 4 with fragment 7 was carried out as described in example 3. After purification by preparative HPLC, the desired adduct (100% purity) was obtained. The yield was 45%.

Analytical grade HPLC (Gemini, Phenomenex, C18, 250x4.6mm, 5 μm; mobile phase: 29% CH)₃Aqueous CN + 0.1% TFA, λ 220 nm). Retention time: 10.84 and 15.22 minutes. Mass of Maldi: 2120.89[ M + H]⁺。

¹H-NMR(DMSO-D₆) Main displacement, δ: 9.94,9.28,9.04,8.58,8.52,8.27-8.17,8.03,7.93-7.73,7.55,7.37,7.25,7.11-7.07,6.82,6.56,6.41，5.90，5.42-5.29，4.95，4.60-4.53，4.46，4.37，4.25，4.16，4.01-3.96，3.84，3.65-3.37，3.17，3.10，3.01-2.88，2.42-2.36，1.90-1.86，1.75-1.71，1.61-1.58，1.50-1.30，0.89。

Example 6

Synthesis of ST5546TF1

The coupling reaction of fragment 4 with fragment 8 was carried out following the procedure described in example 3. The crude reaction product obtained from the cycloaddition reaction was subjected to preparative HPLC (Alltima, Alltech, RP18, 250X22mm, 10 μm; mobile phase: 28% CH)₃Aqueous CN + 0.1% TFA, λ 220 nm). After lyophilization, ST5546TF1 (100% pure) was obtained. The yield was 38%.

Analytical grade HPLC (Gemini, Phenomenex, C18, 250x4.6mm, 5 μm; mobile phase: 28% CH₃Aqueous CN + 0.1% TFA, λ 220 nm). Retention time: 11.38 and 16.16 minutes. Mass of Maldi: 2480[ M + H ]]⁺。

¹H-NMR(D₂O) main shift, δ: 8.73,8.52,7.83-7.74,7.62,7.39,7.19,7.05,6.93,6.87,6.63,5.58-5.49,4.91,4.68-4.26,4.04,3.85-3.42,3.24-3.12,2.93-2.87,2.77,2.65-2.60,2.11,1.93,1.82,1.72,1.63,1.58-1.49,1.12.

Example 7

Synthesis of ST5744TF1

Mixing sodium ascorbate (0.014 mmol) and CuSO₄·5H₂O (0.0014 mmole) in water (14. mu.l) was added to (DMF/H) containing fragment 9(15mg, 0.014 mmole) and fragment 10(34mg, 0.016 mmole)₂O: 1/1, 2ml) in water. The resulting reaction mixture was stirred at room temperature for 1.5 hours. The solvent was subsequently removed under reduced pressure. By passing through HPLC (column, Alltima, Alltech, C18, 10 μm, 250X22 mm; mobile phase: 30% CH)₃Aqueous CN + 0.1% TFA) to afford the desired adduct. The yield was 37%.

Analytical grade HPLC (column Gemini, mobile phase 29% CH)₃Aqueous CN + 0.1% TFA). Retention time: 9.2 and 12.6 minutes. Maldi-TOF: [ M + H ]]⁺2988.78。

¹H-NMR(DMSO-D₆+D₂O) main shift, δ: 9.30,8.56,8.40,8.22,8.19,8.01,7.92-7.85,7.83,7.78-7.69,7.53,7.37,7.23,7.08,6.68,5.42-5.3,5.21,5.10,4.93,4, 74,4.37-4.34,4.23,4.20-4.03,3.89,3.85,3.61,3.56-3.36,3.29-3.16,3.07,3.00-2.73,2.38,2.10,1.85,1.72,1.55,1.40-1.30,1.23,0.87.

Example 8

Synthesis of ST5745TF1

Mixing sodium ascorbate (0.016 mmol) and CuSO₄·5H₂O (0.0016 mmole) in water (16. mu.l) was added to (DMF/H) containing fragment 11(33.2mg, 0.032 mmole) and fragment 12(84mg, 0.031 mmole)₂O: 4/3, 3.5ml) in water. The resulting solution was irradiated with microwaves (90W) for 2 minutes. A maximum temperature of 120 ℃ was observed. By passing through HPLC (column, Alltima, Alltech, C18, 10 μm, 250X22 mm; mobile phase: 32% CH)₃Aqueous CN + 0.1% TFA) to afford the desired adduct (97% purity). The yield was 42%.

Analytical grade HPLC (column Gemini, mobile phase 29% CH)₃Aqueous CN + 0.1% TFA). Retention time: 10.2 and 12.5 minutes. Mass of Maldi: [ M + H ]]⁺3723。

¹H-NMR(DMSO-D₆+D₂O) main shift, δ: 9.05,8.34-8.09,7.82-7.71,7.42-7.24,7.06-6.99,6.66,5.49,5.55-5.11,4.79,4.57,4.37-3.97,3.70-3.38,3.16,3.01-2.87,2.34-2.32,2.00-1.55,1.42-1.28,1.19,0.84.

Example 9

Synthesis of fragment 1

c{Arg-Gly-Asp-D-Phe-Amp[CO-CH₂-(O-CH₂-CH₂)₂-O-CH₂-CO-N₃]}

Microwave-assisted solid phase synthesis of cyclopeptide acylhydrazide

Fmoc-Gly-SASRIN(2.53g, 2 mmol) were suspended in a solution of 20% piperidine in DMF (40ml) and irradiated (25W) for 3 min. After filtration and washing of the resin, a solution containing the next amino acid (2 equivalents) was added followed by a further DMF (36ml) solution containing 2 equivalents of HOBT and TBTU. Finally, DIPEA (4 equiv) dissolved in NMP (5ml) was added and the suspension was irradiated at 30W for 5 min. After filtration and Fmoc deprotection, the next coupling reaction was performed in the same manner until the peptide was complete. The order of addition of the amino acids was Fmoc-Arg (Pmc) -OH, Fmoc-Amp building block (building block) (see FIG. 3a for synthesis), Fmoc-D-Phe-OH and Fmoc-Asp (OtBu) -OH.

After final Fmoc deprotection and washing, cleavage from the resin was achieved by treatment with 1% TFA in DCM (60ml) for 15 min. After filtration, the same operation was repeated 5 times. The combined filtrate was neutralized by adding pyridine and brought to a dry state. HOBT and TBTU (3 equiv.) and 1% DIPEA were added to dissolve in CH₃CN (1500ml) and the reaction mixture was stirred at room temperature for 1 hour. The solvent was then evaporated under reduced pressure. After purification by flash chromatography (DCM/MeOH: 94/6 → 92/8), the desired protected cyclic peptide was obtained (50% yield).

The latter is dissolved in TFA/H₂O: 95/5 and stirred at room temperature for 1 hour. The solvent was then evaporated under reduced pressure and passed through from TFA/Et₂The cyclic peptide was obtained after purification by precipitation with O (98% yield).

Analytical grade HPLC (column, Purosphere STAR)Merck, RP18, 250x4mm, 5 μm; mobile phase: 20% CH₃Aqueous CN + 0.1% TFA; λ 220 nm). Retention time 9.14 minutes. Mass of Maldi-Tof: 870.13[ M + H]⁺。

The deprotected acylhydrazide (0.32 mmol) and HOAT (1.91 mmol) were dissolved in DMF (7ml) and tert-butyl nitrite (0.38 mmol) was added. The reaction mixture was stirred for 30 minutes. The acyl azide was used in the next step without isolation and without any purification.

Example 10

Synthesis of fragment 2

HCl.Ala-Cit-PABC-CPT

Step 1

A solution of Boc-Cit-OH (1g, 3.63 mmol), PABA (1.3g, 10.9 mmol), HOAT (0.74g, 5.45 mmol), DIPEA (0.93ml, 5.45 mmol) and DCC (1.12g, 5.45 mmol) in DMF (65ml) was stirred at room temperature overnight. After evaporation of the solvent under reduced pressure, the residue was purified by flash chromatography (DCM/MeOH: 90/10 → 85/15). By reacting the previous intermediate with TFA/DCM: 1/1 reaction for Boc deprotection followed by solvent removal under reduced pressure gave TFA. Cit-PABA (520 mg). The yield was 73%.

Step 2

To a mixture of Alloc-Ala-OH (472mg, 2.68 mmol), DCC (272mg, 1.34 mmol) and DIPEA (460 μ l, 2.68 mmol) at 0 ℃ in DCM/DMF (v/v. 1/1, 20ml) was added tfa. cit-PABA and the solution was stirred for 6 h. The solvent was removed under reduced pressure and the residue was dissolved in water at pH 2. The resulting solution was extracted 2 times with EtOAc. By adding NaHCO₃The aqueous phase was neutralized and the water was removed under reduced pressure. Purification by scintillation chromatography (EtOAc/MeOH ═ 85/15) yielded Alloc-Ala-Cit-PABA (398 mg). The yield was 69%.

Step 3

4-Nitro-phenylchloroformate (36 μ l) dissolved in DCM (20ml) and pyridine (150 μ l)3mg, 1.8 mmol) was added to a solution of the latter (392mg, 0.9 mmol) in dry DMF (5ml) and the reaction mixture was stirred for 1 hour. The solvent was removed under reduced pressure and the residue was ice-cooled to Et₂And O grinding for several times.

Step 4

7- (2-Aminoethoxyimine) -methyl-camptothecin HCl (423.5mg, 0.90 mmol) and TEA (150. mu.l, 1.1 mmol) were added to a DMF (25ml) solution of the previous adduct and the reaction mixture was stirred for 5 hours. The solvent was removed under reduced pressure and the residue was triturated with water several times to remove excess TEA. After purification by flash chromatography (DCM/MeOH: 90/10), protected fragment 2(320mg, 0.36 mmol) was obtained. The yield was 40% (2 steps).

Step 5

Bu is put into₃SnH (220. mu.l, 0.8 mmol) in DCM (3.8ml), water (40. mu.l) and finally Pd [ (PPh)₃]₄(17mg, 0.014 mmol) was added to a DMF (3.8ml) solution of protected fragment 2 obtained above and the resulting reaction mixture was stirred for 15 minutes. The solvent was removed under reduced pressure to give a solid which was taken up in water (65ml) at pH 3. Subjecting the aqueous layer to Et₂O (25mlx3) extraction followed by concentration gave pure fragment 2 as the hydrochloride salt. The yield was 93%.

HPLC (Gemini, Phenomenex, C18, 250X4.6mm, 5 μm; mobile phase: 28% CH₃Aqueous CN + 0.1% TFA, λ 220 nm). Retention time: 8.9 and 12.3 minutes. Mass of Maldi: 834[ M + Na]⁺。

Example 11

Synthesis of fragment 3

TFA.Phe-Lys(Alloc)-PABC-CPT

Following the procedure described in example 10, starting from Boc-Lys (Alloc) -OH instead of Boc-Cit-OH and using Boc-Phe-OH instead of Alloc-Ala-OH in step 2, the target compound was obtained.

Analytical grade HPLC (Purosphere STAR, M)erck, 5 μm; mobile phase: 35% CH₃Aqueous CN + 0.1% TFA, λ 220 nm). Retention time: 18.00 and 25.29 minutes. Mass of Maldi: 965[ M + Na ]]⁺。

Example 12

Synthetic fragment 4(HC ≡ C-CO-Ala-Cit-PABC-CPT)

DIPEA (0.31 mmol), propiolic acid (0.18 mmol) and HOAT (0.18 mmol) were added to a solution of fragment 2(0.12 mmol) in DMF (3ml) and the solution was allowed to cool at 0 ℃ followed by DCC (0.21 mmol). The reaction mixture was stirred at room temperature for 1.5 hours. After removal of the solvent under reduced pressure, the residue was purified by flash chromatography (DCM/MeOH: 9/1 → 8/2). The yield was 72%.

Analytical grade HPLC (Gemini, Phenomenex, C18, 250x4.6mm, 5 μm; mobile phase: 31% CH₃Aqueous CN + 0.1% TFA, λ 220 nm). Retention time: 11.46 and 16.14 minutes. Mass of Maldi: 863.8[ M + H]⁺And 885.8[ M + Na ]]⁺。

Example 13

Synthesis of fragment 5c { Arg-Gly-Asp-D-Phe-Amp- [ CO- (CH)₂)₂-(O-CH₂-CH₂)₂-O-(CH₂)₂-N₃]}

Following the procedure described in example 9, the building group Fmoc-Amp [ CO- (CH) was incorporated in step 2 of SPPS₂)₂-(O-CH₂-CH₂)₂-O-(CH₂)₂-N₃]To synthesize a target cyclic peptide.

Analytical grade HPLC (Gemini, Phenomenex, C18, 250x4.6mm, 5 μm; mobile phase: 30% CH₃Aqueous CN + 0.1% TFA, λ 220 nm). Retention time 8.3 minutes. Mass of Maldi: 881[ M + H]⁺。

Example 14

Synthesis of fragment 6c { Arg-Gly-Asp-D-Phe-Amp- [ CO- (CH)₂)₂-(O-CH₂-CH₂)₂-O-(CH₂)₂-NH-Cit-CO-(CH₂)₂-(O-CH₂-CH₂)₂-O-(CH₂)₂-N₃]}

Following the procedure described in example 9, the building block Fmoc-Amp- [ CO- (CH) was incorporated in step 2 of SPPS₂)₂-(O-CH₂-CH₂)₂-O-(CH₂)₂-NH-Cit-CO-(CH₂)₂-(O-CH₂-CH₂)₂-O-(CH₂)₂-N₃]Replacement of Fmoc-Amp [ CO-CH ]₂-(O-CH₂-CH₂)₂-O-CH₂-CO-N₃]And synthesizing the cyclic peptide.

Analytical grade HPLC (Gemini, Phenomenex, C18, 250x4.6mm, 5 μm; mobile phase: 25% CH₃Aqueous CN + 0.1% TFA). Retention time 10.79 minutes. Mass of Maldi-Tof: 1241[ M + H]⁺。

Example 15

Synthesis of fragment 7

c{Arg-Gly-Asp-D-Tyr-Amp[CO-(CH₂)₂-(O-CH₂-CH₂)₂-O-(CH₂)₂-NH-Cit-CO-(CH₂)₂-(O-CH₂-CH₂)₂-O-(CH₂)₂-N₃]}

Following the procedure described in example 14, Fmoc-D-Tyr- (t-Bu) -OH was incorporated in step 3 of SPPS to synthesize the target cyclic peptide.

Analytical grade HPLC (Gemini, Phenomenex, C18, 250x4.6mm, 5 μm; mobile phase: 22% CH)₃Aqueous CN + 0.1% TFA, λ 220 nm). Retention time 8.87 minutes. Mass of Maldi: 1256.96[ M + H]⁺。

¹H-NMR(D₂O) main shift, δ: 7.43,7.29,7.19,6.94,4.93,4.59,4.53-4.37,4.01-3.65,3.57,3.35,3.27,3.14-3.07,2.95-2.87，2.79-2.72，2.03-1.60。

Example 16

Synthesis of fragment 8

c{Arg-Gly-Asp-D-Tyr-Amp[CO-(CH₂)₂-(O-CH₂-CH₂)₂-O-(CH₂)₂NH-Cit]₂-CO-(CH₂)₂-(O-CH₂-CH₂)₂-O-(CH₂)₂N₃}

Following the procedure described in example 15, Fmoc-Amp- [ CO- (CH) was incorporated in step 2 of SPPS₂)₂-(O-CH₂-CH₂)₂-O-(CH₂)₂NH-Cit]₂-CO-(CH₂)₂-(O-CH₂-CH₂)₂-O-(CH₂)₂N₃To synthesize a target cyclic peptide.

Analytical grade HPLC (Gemini, Phenomenex, C18, 250x4.6mm, 5 μm; mobile phase: 21% CH₃CN, λ 220 nm). Retention time 11.62 minutes. Mass of Maldi: 1617.31[ M + H]⁺。

¹H-NMR(D₂O), main shift, δ: 7.23,7.10,7.05,6.73,4.58,4.40,4.33-4.17,3.82-3.47,3.40,3.38,3.16-3.05,2.96-2.82,2.75,2.69,2.58,1.84-1.40.

Example 17

Synthesis of fragment 9

Step 1

DCC (97.2mg, 0.47 mmol), p-nitrophenol (437mg, 0.31 mmol), TEA (1.31ml, 9.43 mmol) and DMAP (7.7mg, 0.06 mmol) were added to a suspension of anhydrous 3, 6, 9-trioxaundecanedioic acid (2.1g, 9.43 mmol) in DCM (63ml) at 0 ℃. After 30 minutes, the reaction mixture was washed with water, 0.1N HCl and water and dried over sodium sulfate, then concentrated to a small volume and placed in a refrigeratorAfter 1 hour, the mixture is filtered. Propargylamine hydrochloride (144mg, 1.57 mmol) and TEA (262 μ l, 1.88 mmol) were added to the filtrate and the solvent was removed under reduced pressure after several minutes. The resulting residue was dissolved in water (20ml) and filtered through Dowex 50W X8. The mother liquor was extracted 2 times with DCM to remove residual nitrophenol and after concentration the desired alkyne-PEG-CO was generated as a white solid₂H. The yield was 72%.

Step 2

DCC (25mg, 0.12 mmol) was added to Ala-Cit-PABC-CPT (fragment 2, 56mg, 0.06 mmol), alkyne-Peg-CO₂H (22mg, 0.085 mmol), HOAT (16mg, 0.12 mmol) and DIPEA (41. mu.l, 0.24 mmol) in DMF (1.5ml) in ice cold (0 ℃ C.). The reaction mixture was then stirred at room temperature overnight. After filtration, the filtrate was concentrated to dryness and the resulting residue was purified by flash chromatography (DCM/MeOH: 85/15) to finally give the desired adduct as a yellow solid (40 mg). The yield was 63.5%.

Analytical grade HPLC (column Gemini Phenomenex C18; 250X4.6mm, 5 μm; 32% CH)₃Aqueous CN + 0.1% TFA). Retention times 11.6 and 16.3 minutes. ESI mass: [ M + H ]]⁺1053.42。

Example 18

Synthesis of fragment 10 (see FIG. 3e)

Step 1

DCC (84mg, 0.41 mmol) was added to an ice-cold (0 ℃ C.) solution of L-glutamic acid di-tert-butyl ester hydrochloride (100mg, 0.34 mmol), azidoacetic acid (41mg, 0.41 mmol), HOAT (0.41 mmol) and DIPEA (127ml, 0.74 mmol) in DCM (4.6 ml). The reaction mixture was stirred at room temperature for 2.5 hours. After filtration, the organic solution was diluted to 30ml with DCM and washed with water, 1N HCl, 5% NaHCO₃And water washing. The solvent was removed under reduced pressure and the resulting residue was dissolved in TFA (3ml) and stirred for 1 hour. TFA was subsequently removed under reduced pressure to yield 2- (2-azido-acetylamino) -glutaric acid.

Step 2

2- (2-azido-acetylamino) -glutaric acid was dissolved in a DCM/DMF (8/1, 45ml) mixture. Standard coupling reactions with tert-butyl-12-amino-4, 7, 10-trioxadecanoate (281mg, 1.01 mmol), HOAT (137mg, 1.01 mmol), DIPEA (174. mu.l) and DCC (209mg, 1.014 mmol) gave a crude product which was purified by scintillation chromatography (DCM/MeOH: 95/5) to give the desired biscarboxylate intermediate (175mg) as a solid product. The yield was 68.4%.

¹H-NMR(CDCl₃)δ：7.54，7.23，6.74，4.42，4.01，3.70，3.61，3.41，2.50，2.35，2.08，1.44。

Step 3

The compound obtained above was deprotected using TFA under standard conditions. Once all starting material disappeared, TFA was removed under reduced pressure to give the dicarboxylic acid intermediate, which was used in the next step without any further purification.

Step 4

A solution of N-hydroxysuccinimide (63mg, 0.55 mmol) in DMF was added to a solution of the intermediate product obtained above at 0 deg.C followed by DCC (115mg, 0.55 mmol). The reaction mixture was stirred at room temperature overnight. After standard operation the crude desired product is obtained, which is used in the next step with any further purification.

Step 5

The intermediate obtained above was dissolved in DCM (2ml) and reacted with the cyclic peptide dissolved in DMF (3.5ml) in the presence of DIPEA (153. mu.l, 0.93 mmol) at room temperaturec{Arg(Pmc)-Gly-Asp(OtBu)-D-Tyr(tBu)-Amp}(725mg, 0.69 mmol) for 1.5 hours. The cyclic peptidec{Arg(Pmc)-Gly-Asp(OtBu)-D-Tyr(tBu)-Amp}Is passed through SPPS according to the procedure described in example 15 and Fmoc-Amp- [ CO- (CH) is replaced by Fmoc-Amp (Cbz) -OH₂)₂-(O-CH₂-CH₂)₂-O-(CH₂)₂-NH-Cit-CO-(CH₂)₂-(O-CH₂-CH₂)₂-O-(CH₂)₂-N₃]Is prepared. The crude residue was prepared by preparative HPLC (column Alltima, C18 Alltech; 10 μm, 250X22 mm; 69% CH)₃Aqueous CN + 0.1% TFA). The yield was 48%.

Step 6

Final deprotection reaction in DCM (1mL) by using TFA (540 equiv.) and thioanisole (110 equiv.) to give the crude product via cooling from ice with Et₂O was purified by several successive precipitations. The desired fragment 10 was obtained as a white solid. The yield was 69%.

Analytical grade HPLC (column Gemini Phenomenex C18; 250X4.6mm, 5 μm; 22% CH)₃Aqueous CN + 0.1% TFA). Retention time 10.9 minutes. MALDI mass: [ M + H ]]⁺1935.22。

Example 19

Synthesis of fragment 11

The succinimide derivative (step ii) during the synthesis of the building block from fragment 5, dissolved in DCM (0.5ml) (fig. 3c, 37mg, 0.11 mmol) was added to a solution of fragment 2(80mg, 0.094 mmol) and DIPEA (19 μ l, 0.11 mmol) in DMF (1 ml). The reaction mixture was stirred at room temperature for 5 hours. After evaporation of the solvent under reduced pressure, the residue was subjected to preparative HPLC (column Alltima, 10 μm, 250X22 mm; mobile phase: 37% CH)₃Aqueous CN + 0.1% TFA). Retention times 9.7 and 12.4 minutes. The yield was 76.3%. ESI mass: [ M + H ]]⁺1041.42。

Example 20

Synthesis of fragment 12

Step 1

Mixing sodium ascorbate (90 μ l) and 0.5M CuSO₄·5H₂O (45. mu.l) in 2.5M aqueous solution was added to (1, 3-di-prop-2-ynylcarbamoyl-propyl)Benzyl carbamate (72.5mg, 0.20 mmol) and c { Arg (Pmc) -Gly-Asp (OtBu) -D-Tyr (tBu) -Amp- [ CO- (CH)₂)₂-(O-CH₂-CH₂)₂-O-(CH₂)₂-N₃]} (572mg, 0.45 mmole) in DMF/water (7/5, 12 ml). The above cyclic peptide was synthesized according to the method described in example 13 using Fmoc-D-Tyr- (t-Bu) -OH instead of Fmoc-D-Phe-OH. The resulting reaction mixture was irradiated with microwaves (90W) for 2 minutes. A maximum temperature 121 is observed. This irradiation was repeated 3 times until complete disappearance of the starting materials by HPLC (column Gemini, 250X4.6mm, 5 μm; mobile phase: 35% CH)₃Aqueous CN + 0.1% TFA). The solvent was removed under reduced pressure and the crude reaction mixture was purified by flash chromatography (DCM/MeOH gradient: 93/7 → 90/10 → 80/20) to yield the desired product (417 mg). The yield was 71%. ESI mass: 1453.6(m/z 2+), 969.4(m/z 3 +).

Step 2

The above-obtained product (406mg), dissolved in a mixture of DMF (3ml) and MeOH (5ml), was deprotected by the action of ammonium formate (44mg, 0.70 mmol) and Pd/C (200mg) to remove the benzyloxycarbonyl protecting group. The suspension was stirred for 3 hours and then filtered. The solvent was removed under reduced pressure and the product formed was used in the next step without any further purification.

Step 3

A solution of the product obtained above in DMF (3ml) was added to a solution of intermediate from a standard coupling reaction of propargylglycine and methyl- (PEG)12-NHS (102mg, 0.15 mmol) in DCM (4.5ml), followed by addition of HCTU (62mg, 0.15 mmol) and DIPEA (51. mu.l, 0.30 mmol). The resulting solution was stirred at room temperature for 2 hours. After removal of the solvent under reduced pressure, the residue was dissolved in DCM (300ml) and washed with water. The organic phase was then evaporated to yield the desired adduct (312 mg). The yield was 67%. ESI mass: 1741(m/z 2+), 1168(m/z 3 +).

Step 4

The intermediate obtained above was fully deprotected by using a mixture of TFA/DCM/thioanisole (1/1/0.3). By cooling with ice Et₂Precipitation in O to purify the compound to yield the desired fragment 12(245 mg). The yield was 92%. MALDI mass was found: 2679.79.

biological test results

Conjugates with integrin receptor alpha_vβ₃And alpha_vβ₅Solid phase binding assay of

Receptor binding assays were performed as described in Orlando r.a., et al, j.biol.chem.1991, 266, 19543. Alpha is alpha_vβ₃And alpha_vβ₅In coating buffer (20mM Tris, pH 7.4, 150mM NaCl, 2mM CaCl), respectively₂，1mM MgCl₂，1mM MnCl₂) Was diluted to 500ng/ml and 1. mu.g/ml and portions (100. mu.l) were added to 96-well microtiter plates and incubated overnight at 4 ℃. The plate was passed through blocking/binding buffer (50mM Tris, pH 7.4, 100mM NaCl, 2mM CaCl)₂，1mM MgCl₂，1mM MnCl₂1% bovine serum albumin) was washed 1 time and then incubated at room temperature for an additional 2 hours. The disc is washed 2 times with the same buffer and is contacted with the radiolabeled ligand [ 2 ] at room temperature in the presence of a competitive inhibitor¹²⁵I]Lepidemic inhibitor (Echistatin) (Amersham Pharmacia Biotech, 0.05 nM; p.alpha._vβ₅0.1nM) was incubated for 3 hours. After this incubation, wells were washed and radioactivity was measured using a gamma counter (Packard). Nonspecific binding of ligands was determined using a molar excess (200nM) of icilodescale.

Calculating the ICs shown in tables 1 and 2₅₀Values were taken as the concentration of compound required to inhibit desmopressin binding by 50% and were estimated by the Prism GraphPad program. Ki values for competing ligands were calculated according to the Cheng-Prusoff equation (Cheng Y.C., et al, biochem. Pharmacol., 1973, 22, 3099). These values are the mean ± log standard error of triplicate determinations from two independent experiments.

Most of these conjugates exhibited potent inhibitory activity in the low nanomolar (nanomolar) range. It is noteworthy that the in vitro activity demonstrated by ST3280 is based mainly on the intrinsic activity of the decomposition products due to the instability of the compound itself.

TABLE 1

Inhibition [ 2 ]¹²⁵I]Lepidemic inhibitor and alpha_vβ₃Receptor binding

Compound (I)	IC50±log SE(nM)	Ki(nM)
			Lepidemic inhibitor	0.28±0.08	0.26
ST3833	78.4±1.5	61.0
			ST3280	9.7±0.06	8.5
ST4167	11.0±0.8	8.7
			ST5744TF1	3.01±0.11	2.4
ST5745TF1	6.21±0.09	4.92

TABLE 2

Inhibition [ 2 ]¹²⁵I]Lepidemic inhibitor and alpha_vβ₅Receptor binding

Compound (I)	IC50±log SE(nM)	Ki(nM)
			Lepidemic inhibitor	0.29±0.02	0.33
ST3833	87.8±1.21	68.2
			ST3280	34.4±0.8	23.0
ST4167	18.4±0.89	13.8
			ST5744TF1	3.84±0.12	2.95
ST5745TF1	3.15±0.11	2.41

Adhesion assay of tumor cells to vitronectin

A2780 human ovarian and PC3 prostate cancer cells were grown in medium RPMI 1640 containing 10% fetal bovine serum and 50. mu.g/ml gentamicin sulfate. The cells were maintained at saturated humidity and 95% air and 5% CO₂In a 37 c incubator in a gaseous environment. A2780 tumor cell strain expresses high amount of alpha_vβ₅Integrins, while PC3 expresses low amounts of alpha_vβ₃And alpha_vβ₅Integrins.

In a 96-well tissue culture plate, 50. mu.l/well of vitronectin (5. mu.g/ml) solution was added for 2 hours at room temperature. The plates were inverted to remove the solution. 50 μ l/well of 1% BSA solution was added for 1 hour at room temperature. These plates were washed by adding 100. mu.l/well of RPMI 1640 medium without Fetal Calf Serum (FCS). This washing was repeated 2 times. Different concentrations of these molecules were added between 0.039 and 20 μ M. These solutions were prepared by dilution in a 1: 2 ratio in medium without FCS. Tumor cells in saline solution in flasks were rinsed by adding 5ml of medium without FCS and 1% BSA before peeling with a spatula. Tumor cells were counted after resuspension and added at appropriate cell densities (40000 and 50000 cells/well). The plates were placed in a humidified incubator (37 ℃, 5% CO)₂) By incubation 1And (4) hours. Subsequently, the plates were inverted to remove the solution and passed through 200. mu.l/well of Ca-containing solution²⁺And Mg²⁺Washed 1 time with PBS solution. Tumor cells were fixed with 4% paraformaldehyde (100. mu.l) solution in 0.2M Sorensen phosphate buffer, pH 7.2-7.4, for 10 min at room temperature. The plates were poured over and 1% Toluidine blue (Toluidine Blu) solution (100. mu.l) was added at room temperature for 10 min. The trays were rinsed 2 times by immersion in redistilled water and allowed to dry in a 60 ℃ incubator (Kottermann). 100 μ l/well of 1% SDS was added. The plates were left stirring for 20 minutes at room temperature and then evaluated at 600nm by a Victor 1420 multiple labeling counter (Wallac).

Evaluation of IC as a parameter for measuring the efficacy of these molecules in the inhibition of tumor cell adhesion to vitronectin Using the "ALLFIT" computer program₅₀The value is obtained.

The conjugates studied were found to be able to function as IC₅₀Values between 0.39 and 4.6 μ M (Table 3) and did not show an over-selective manner for tumor cell lines to block the adhesion of tumor cells (PC3 and A2780) to extracellular matrix components such as vitronectin, cell surface receptor integrin α_vβ₃And alpha_vβ₅The ligand of (1). E.g. for alpha_Vβ₃Binding affinity of the receptor, ST3280 vs. alpha_Vβ₅The activity of the receptor is the result of cleavage of the compound rather than the compound itself.

TABLE 3

Anti-adhesion efficacy of the conjugate against vitronectin adhesion to A2780 ovarian cancer cells and PC3 prostate cancer cells (1 hour treatment)

Cytotoxicity of conjugates against different tumor cell lines

Amine thiocyanate B (sulforhodamine B) assay was used to evaluate the efficacy of compounds on surviving cells. PC3 human prostate cancer and a2780 human ovarian cancer cells were used to measure the efficacy of the compounds on cell growth. A2780 and PC3 tumor cells were grown in medium RPMI 1640(GIBCO) containing 10% fetal bovine serum.

Tumor cells were seeded in 96-well tissue culture plates at about 10% confluence and allowed to adhere and recover for at least 24 hours. Different concentrations of drug were then added to each well to calculate their IC₅₀Value (i.e., concentration that inhibits cell survival by 50%). The plates were incubated at 37 ℃ for 72 hours. At the end of the treatment, the plates were rinsed by removing the supernatant and adding PBS3 times. PBS solution (200. mu.l) and ice-cold 80% trichloroacetic acid (TCA) (50. mu.l) were added. The plates were incubated on ice for at least 1 hour. The TCA was removed and the discs were rinsed 3 times by immersion in distilled water and dried on paper at 40 ℃ for 5 minutes. Then a solution of 0.4% thiocyanoamine B in 1% acetic acid (200. mu.l) was added. The plates were incubated at room temperature for an additional 30 minutes. The thiocyanamide B was removed, the discs rinsed by immersion 3 times in 1% acetic acid and dried on paper at 40 ℃ for 5 minutes. Tris (10mM, 200. mu.l) was then added and the plates were kept under stirring for 20 minutes. Cell survival was measured by optical density and using a Multiskan fluorescence spectrophotometer (wavelength 540 nm). The number of killed cells was calculated as the percentage reduction in the binding of thiocyanamide B compared to control cultures.

These ICs₅₀Values were calculated using the "ALLFIT" program.

The antiproliferative activity of the 3 conjugates was compared by two human tumor cell lines, i.e. a2780 ovarian tumor cells expressing high amounts of integrin and PC3 prostate tumor cells expressing low amounts of integrin. As shown in Table 4, these molecules display IC on tumor cells₅₀Significant cytotoxic efficacy with a value of 8 nM. All these conjugates showed minimal effect on PC3 tumor cells expressing low amounts of Integrin (IC)₅₀The value is between 1 and 4.6. mu.M). In particular, the 3 compounds showed more specific anti-proliferative efficacy on a2780 tumor cells compared to PC3 tumor cells (table 4) and on a2780 tumorsThe cells are about 100 times more potent than the latter.

TABLE 4

Cytotoxicity of the conjugate against A2780 ovarian cancer cells and PC3 prostate cancer cells (72 hr treatment)

In vivo evaluation of the antitumor Activity of conjugate ST3833 on tumor growth of xenografted ovarian cancer in CD1 nude mice

Tumor cell line (3x 10)⁶) Was injected subcutaneously into the right flank of CD1 nude mice (Harlan). Each experimental group included 10 mice. Tumors were planted on day 0 and tumor size was measured with a Vernier caliper every two weeks after tumor growth. According to the formula TV (mm)³)＝d²x D/2 the tumor volume is calculated where D and D are the shortest and longest diameters, respectively. On day 3 after tumor inoculation, drug treatment was started when the tumor was just measurable. Drugs were administered subcutaneously for two weeks according to the course of treatment qd x5/w x2w at various doses of volume 10 ml/kg. Control mice were treated with vehicle (10% DMSO).

Drug efficacy was evaluated by the following manner.

a) TVI of drug-treated mice versus control mice is expressed as follows: TVI (%) ═ 100 (mean TV value of treated mice/mean TV value of control mice) x 100. After the final treatment, TVI was evaluated for 6 days, which time corresponded to the time required to observe doubling of tumor volume in control mice.

b) According to the formula: log Cell Kill (LCK) values were calculated for (T-C)/3.32x DT, where T and C are the mean time (days) required for the tumor distribution to reach the determined volume for the treated (T) and control (C) groups, respectively, and DT is the time required to observe tumor volume doubling in the control mice.

c) CR is defined as the disappearance of the tumor lasting for at least 6 days after the end of the treatment. Tumors that did not regrow by the end of the experiment are considered "cured".

The toxic effects of drug treatment were evaluated in the following manner.

a) BWL was calculated according to the formula BWL (%) -100- (xth balance body weight/xth balance body weight 1) x100, where day 1 is day 1 of treatment and day x is any day thereafter. The highest (maximum) BWL values are reported in the table. Mice were weighed daily throughout the treatment period.

b) Lethal toxicity is defined as the death of any treatment group of mice that occurs prior to the death of any control group of mice. Mice were investigated daily for mortality. TI (therapeutic index) was calculated as the ratio MTD/ED 80.

Results

In xenografted CD1 nude mice, the antitumor activity of ST3833 was the most potent in vitro reactivity against tumors. The molecule appeared to be administered subcutaneously at about the Maximum Tolerated Dose (MTD) of 25mg/kg according to this course of treatment qd x5/w x2w, since the BWL value was 25% and 1 out of 10 mice died. ST3833 showed potent antitumor efficacy because ST3833 produced complete regression of all tumors (cured mice were 100% at this MTD on day 90) (table 5). 50% of the mice cured were observed at 1/3MTD (8.3 mg/kg). At lower doses (2.77 and 0.92mg/kg), the cured mice were up to 30%. The persistence of the efficacy after the last treatment and the good tolerability of the conjugate showed a high therapeutic index (TI ═ 8.9), which suggests a high therapeutic efficacy of the conjugate.

TABLE 5

Subcutaneously administered (qdx5/wx2w) ST3833 antagonized the antitumor activity of xenografted A2780 ovarian cancer in CD1 nude mice

^aSubcutaneous dose per administration

^bMaximum BWL% due to drug treatment

^cDeath/treatment animals

^dTVI% for control mice

^eCR: tumor disappearance is at least 10 days

^fAnd (3) curing: mice that were not lesioned 90 days after tumor injection

^gLCK, see methods

^hTI: therapeutic index (MTD/ED80)

In vivo evaluation of the anti-metastatic activity of the conjugate ST3833 against bone metastases resulting from intracardiac injection of PC3 human prostate cancer

Male CD1 nude mice were anesthetized by intraperitoneal injection of 4ml/kg of a mixture (thiamazine: ketamine (ketavet) 100). Intracardiac injection (1X 10) with a 27-gauge needle⁵Cells/0.1 ml/mouse) PC3 tumor cells were inoculated into the left ventricle of the heart of mice. Mice were divided into the following experimental groups (11 mice/group) and 3 days after injection from the tumor, molecules were administered as follows:

vehicle (DMSO 10%) intravenous q4dx4

ST 383356 mg/10ml/kg intravenous q4dx4

To evaluate the antitumor activity of the drug, high resolution whole body radiology was performed using the Faxitron system. Radiologic analysis was performed 30 days after tumor injection. Body weight was recorded and the number of deaths noted throughout the study period.

The conjugate showed good tolerability at intravenous administration of 56mg/kg (q4dx4) since no lethal toxic weight loss was found. The molecule appeared to significantly increase the lifespan by 45% (P < 0.001) and reduce the occurrence of osteolytic injury (from 91% in vehicle-treated mice to 45% in drug-treated mice; Table 6).

TABLE 6

The intravenous administration (q4dx4) of conjugate ST3833 antagonized the anti-metastatic activity of PC3 prostate cancer xenografted in CD1 nude mice

^aIntravenous dose per administration

^bMaximum BWL% due to drug treatment

^cDeath/treatment animals

^dOccurrence of osteolytic injury (30 days after tumor injection, number of metastases from mice in drug-treated group vs. mice in vehicle-treated group)

^eMST: mean time to live

^fILS%: increased life span

P < 0.001 vs vehicle treatment groups (Mann-Whitney test)

Claims (14)

1. Cyclic peptide of formula I

[(L-D)_nE]_m-F-D-PI-SI-CT

Formula I

Wherein

L is a cyclic peptide of formula II which recognizes the alpha-integrin receptor

c(R¹-Arg-Gly-Asp-R²)

Formula II

R¹Is Amp, Lys or Aad;

R²is Phe, Tyr or Amp in the R configuration;

d may be the same or different at each occurrence and is absent or is a divalent radical of the formula III

-SP¹-A¹-SP²-A²-SP³-

Formula III

SP¹Is absent or R³-(CH₂)_q-(OCH₂-CH₂)_q-O-(CH₂)_q-R⁴；

R³And R⁴Are identical or different and are absent or are a divalent radical of the formula IV, VIII or IX

q may be the same or different at each occurrence and is independently an integer from 0 to 6;

A¹is a natural or non-natural (L) or (D) amino acid, absent or containing a hydrophilic side chain;

SP²is absent or associated with SP¹The same;

A²is absent or is in combination with A¹The same;

SP³is absent or associated with SP¹The same;

m is 1 or 2;

n is 1 or 2;

e may be the same or different at each occurrence and is Glu, Lys or absent;

f is the same as E or is absent or a histidine analogue of formula X;

wherein the triazole ring is substituted withA D-PI-SI-CT moiety, the carbonyl moiety being attached to a moiety containing L and SP¹Is as defined above;

PI is a natural or non-natural oligopeptide consisting of (L) or (D) amino acids selected from Ala and Cit;

SI is a divalent radical p-aminobenzyloxycarbonyl;

CT represents a cytotoxic group;

their tautomers, geometric isomers, optically active forms (such as enantiomers, diastereomers and racemic forms thereof) and pharmaceutically acceptable salts thereof;

the premise is that:

at least one D should be present; and is

When E is present and Lys, E is linked to the moiety containing the L group via its amino moiety, or when E is present and Glu, E is linked to the moiety containing the L group via its carboxyl moiety.

2. The cyclic peptide of claim 1, wherein CT is a camptothecin derivative, R¹Is Amp or Aad, R²Is selected from Phe, Amp or Tyr.

3. A cyclic peptide according to claim 1 or 2 wherein m-1 and n-1.

4. A cyclic peptide according to claim 1 or 2, wherein m-1 and n-2.

5. Integrin alpha_vβ₃And alpha_vβ₅Use of a cyclic peptide according to any one of claims 1 to 4 with inhibitory properties as a medicament.

6. Use of a cyclic peptide according to claim 5 having an integrin IC of less than 1 μ M₅₀The value is obtained.

7. A pharmaceutical composition containing, as active principle, at least one cyclic peptide according to any one of claims 1 to 4, in admixture with at least one pharmaceutically acceptable excipient and/or carrier.

8. A method of synthesizing a cyclic peptide according to any one of claims 1 to 3 by reacting a compound of formula V

(CT-SI-PI)-NH₂(formula V)

Wherein CT, SI and PI are as described above,

with an azido-containing derivative of the formula VI,

L-(SP¹-A¹-SP²-A²-SP³)-N₃(formula VI)

L, SP therein¹、A¹、SP²、A²And SP³Is as described above and R⁴Is CO, and wherein CT, SI and PI are as described above.

9. A method of synthesizing a cyclic peptide according to any one of claims 1 to 3 by reacting a compound of formula VII

(CT-SI-PI) -CO-C ≡ CH (formula VII)

Wherein CT, SI and PI are as described above

With a compound of the formula VI,

wherein L, SP in the compound of formula VI¹、A¹、SP²、A²And SP³Is as described above, provided that R⁴Is absent.

10. A method of synthesizing a cyclic peptide according to any one of claims 1, 2 and 4 by reacting a compound of formula XI

(CT-SI-PI)-D-NHCH₂-C ≡ CH (formula XI)

Wherein CT, SI, PI and D are as described above,

with a compound of the formula XII,

[(L-D)_nE]_m-COCH₂-N₃(formula XII)

Wherein L, D and E are as described above.

11. A method of synthesizing a cyclic peptide according to any one of claims 1, 2 and 4 by reacting a compound of formula XIII

(CT-SI-PI)-D-N₃(formula XIII)

Wherein CT, SI, PI and D are as described above

With a compound of the formula XIV,

[(L-D)_nE]_m-CO-CH(NHD)CH₂-C ≡ CH (formula XIV)

Wherein L, D and E are as described above.

12. Use of a pharmaceutical composition according to claim 7 for the preparation of a medicament having anti-cancer activity.

13. A method of treating a mammal suffering from an uncontrolled cell growth, invasion and/or metastasis condition comprising administering a therapeutically effective amount of a pharmaceutical composition according to claim 3 or 4.

14. The method of claim 13, which is for the treatment of ovarian and/or prostate cancer.