KR20040030686A - New use or cyclolignans and new cyclolignans - Google Patents

Abstract

The present invention relates to the use of known cyclolignans in which the lactone ring has a trans structure as an inhibitor of the insulin-like growth factor-1 receptor, as well as the novel compounds and the use of these novel compounds. The compounds can be used to treat IGF-IR dependent diseases, especially cancer. In particular, deoxypodophyllotoxins can be used to treat human leukemia.

Description

New uses of cyclolignans and new cyclolignans {NEW USE OR CYCLOLIGNANS AND NEW CYCLOLIGNANS}

Insulin-like growth factor-1 receptor (IGF-1R) plays an important role in protection against the proliferation, apoptosis and transformation of malignant cells. IGF-1R is also important in maintaining the malignant phenotype of tumor cells, which is involved in the process of forming tumor cells' resistance to the action of anticancer drugs. However, it is assumed that IGF-1R is not necessary for normal cell growth.

IGF-1R consists of two identical extracellular alpha-subunits involved in ligand binding, two identical beta-subunits carrying the transmembrane domain, and an intracellular tyrosine kinase domain. As a result of ligand receptor interactions, tyrosine residues in the tyrosine kinase domain are phosphorylated, wherein the domain spans amino acids 973-1229 of the β-subunit. The major sites of phosphorylation are tyrosine clusters located at positions 1131, 1135 and 1136 [LeRoith, D et al., Endocr Rev 1995 April; 16 (2), 143-63]. After autophosphorylation, receptor kinases phosphorylate intracellular proteins such as insulin receptor substrate-1 and Shc, thereby phosphatidyl inositol-3 kinase and mitotic promoter activating protein kinase signaling pathways. Activate each one.

Based on the pivotal role of IGF-1R in malignant cells, it is even more clear that IGF-1R is the target of cancer therapy [Baserga, R., et al., Endocrine vol. 7, no. 1, 99-102, August 1997]. One way to block IGF-1R activity is to induce selective inhibition of the IGF-1R tyrosine kinase. However, selective inhibition of IGF-1R today is not all that easy.

Drugs containing cyclolignan grapephytotoxin have been used for centuries, and attention has been paid particularly to its anticancer properties. However, due to the undesirable side effects of grapephytotoxin, it is being braked for use as an anticancer drug. The cytotoxic mechanism of grapephytotoxin was due to its binding to beta tubulin to inhibit microtubule assembly and mitosis. In order to affect microtubules in the cell free system, it was possible that the concentration was only a few μM. The trans structure of the lactone ring of podophyllotoxin appears to be necessary for binding to beta tubulin. Likewise, its stereoisomer, picropodophyllotoxin, has a cis structure with a lactone ring, and its affinity for microtubules in rats is 50 times lower and LD50 is 35 times higher. Due to the low affinity of picropodophyllotoxin for microtubules, this compound received little attention. In the last few years, interest has been drawn in regard to staphylotoxin derivatives, and therefore it has been considered to use etoposide ie ethylidene glucoside derivatives of 4'-demethyl-epidopodophyllotoxin. Etoposide, which does not affect microtubules, is a DNA topoisomerase II inhibitor and is currently used for cancer therapy. These materials absolutely require 4'-hydroxy instead of 4'-methoxy groups of cyclolignan to inhibit topoisomerase II.

Prior art

A number of synthetic tyrosine kinase inhibitors (called tyrphostin) have been studied by Parrizas, M. et al. (Endocrinology 1997, Vol. 138, No. 4, 1427-1433). The IGF-1R is a member of the tyrosine kinase receptor family and also includes receptors for insulin, epidermal growth factor (EGF), nerve growth factor (NGF) and platelet derived growth factor (PDGF). Since tyrosphostin is very homologous, although two of tyrphostin have weak affinity for IGF-1R, all of typhostine active on IGF-1R crosslink with the insulin receptor. Therefore, it has been suggested that small molecules that can distinguish between the two receptors can be designed and synthesized.

Substrate competitive inhibitors of IGF-1 receptor kinases have been described by Blum, G. et al. (Biochemistry 2000, 39, 15705-15712). A number of major compounds have been reported as inhibitors of isolated IGF-1R kinase. The search for such compounds was aided by knowledge of the three-dimensional structure of the insulin receptor kinase domain (ie, the domain that is 84% homologous to the IGF-1R kinase domain). The most potent of the inhibitors found are Tyrfostin AG538 with an IC50 of 400 nM. However, the inhibitor also blocked insulin receptor kinases.

Kanter-Lewensohn, L et al. [Molecular and Cellular Endocrinology 165 (2000), 131-137] indicate whether tamoxifen (TAM) has a cytotoxic effect on melanoma cells. Revealed that it may be dependent on interference. Although TAM did not have a strong effect on IGF-1 binding and expression of IGF-1R on the cell surface, 15 μM of TAM effectively inhibited tyrosine phosphorylation of IGF-1R beta-subunit.

The relationship between the IGF-1R and the grape filotoxin derivatives has not been previously revealed at all. Literature [The Chemistry of Podophyllum, such as JLHartwell, Fortschritte der Chemie organischer Naturstoffe 15, 1958, 83-166], the podophyllotoxin and different derivatives thereof, as, two plant species that is, grapes pilryum pel tatyum (Podophyllum peltatum) and grape pilryum Derivatives derived from Edo ( Podophyllum emodi ) are described. As mentioned above, the observed cytotoxic effect of podophyllotoxin is due to binding to microtubules and inhibiting mitosis. The same effect of deoxypodophyllotoxins on cells has been described, suggesting why the two compounds and their analogues, the 4′-demethyl analogues, can be used for the treatment of psoriasis (WO 86/04062). ). However, the LD50 of grapephytotoxin in rats was relatively low (14 mg / kg), whereas the LD50 of the deoxy derivatives was strangely higher than 15 times. Other grapephytotoxin derivatives, such as acetylpodophyllotoxin (185 mg / kg) and epipodophyllotoxin (> 200 mg / kg), which were high in LD50 in rats, are thought to be essentially deficient in biological activity [Seidlova- Masinova V. et al., J Nat Cancer Inst, 18, 359-371, 1957].

Evaluation of the structural activity of a number of morpholino derivatives of benzyl-benzodioxol having structural similarity to podophyllotoxin is described in Batra J. et al. [Biochemical Pharmacology, Vol. 35, No. 22, 4013-4018, 1986]. Was performed. Although the compound was tested for its ability to inhibit tubulin polymerization, the morpholino compound most similar to grapephytotoxin was the least active in the series of compounds.

Jurd, L. et al., J. Agric. Food Chem. Vol. 27, No. 5, 1007-1016, 1979, benzyl and cinnamil derivatives of 2,4-di-tert-butylphenol and 1,3-benzodioxol are known to be insect sterilizers.

Object of the present invention

It is an object of the present invention to find new methods and novel compounds for treating IGF-1R dependent diseases, in particular cancer, by inhibiting insulin-like growth factor-1 receptors.

The present invention relates to novel compounds known to inhibit IGF-1R, an insulin-like growth factor-1 receptor for the treatment of IGF-1R dependent diseases, in particular cancer, and novel uses thereof.

1 shows a computer model of a twelve amino acid peptide comprising tyrosine at positions 1131, 1135 and 1136 of the IGF-1 receptor.

Figure 2a shows the structural formulas of the grape filotoxin, deoxypodophyllotoxin compounds, Figure 2b shows the structural formula of epipodophyllotoxin and acetylpodophyllotoxin.

Detailed description of the invention

Three-dimensional structure of a short peptide containing the amino acid sequence of the IGF-1R tyrosine domain comprising tyrosine residues at positions 1131, 1135, and 1136 to find compounds that have the ability to simulate tyrosine residues and interfere with their phosphorylation Was analyzed by a computer program. Then, when 12 amino acid peptides were used, two of the three major tyrosines, that is, tyrosine at positions 1135 and 1136 where IGF-1R should be autophosphorylated for activation, were 0.95 nm (9.5 kPa) between each group. It was found that it is located at a suitable angle of about 60 ° with a space of). The structure of the sequence is shown in FIG. This short distance in the corresponding tyrosine of the insulin receptor has never been observed. 1 also shows the three-dimensional structure of grapephytotoxin and deoxypodophyllotoxin.

Molecular modeling revealed that the inhibitory molecule could consist of two benzene rings separated by only one carbon atom. When two carbon bonds were attempted, the distance between the substituents of the benzene ring was too long (about 1.3 nm; 13 kPa).

Substituents of inhibitors corresponding to hydroxy groups in tyrosine are chosen as methoxy or methylenedioxy groups because they are chemically relatively stable, ie they are not oxidized or phosphorylated. The distance between the substituents should be about 0.95 ± 0.10 nm (9.5 ± 1.0 kPa).

Surprisingly, the two benzene rings that formed angles in some cyclolignans, including podophyllotoxin, were able to simulate two tyrosines, tyrosine at positions 1135 and 1136, almost identically; Suggests that it can inhibit autophosphorylation of residues.

To penetrate the receptor, the inhibitory molecule must be small. For example, the effect of phytotoxin on IGF-1R disappears completely when conjugated to phytophytoxin-4,6-O-benzylidene-β-D-glucopyranoside, which is a glucoside derivative, . In addition, after the lactone ring is reduced to the diol structure, the size of the molecule is increased due to the reduced substituents extending from the molecule, and as a result, the activity of the compound is drastically reduced. Increasing the size by forming methylenedioxy derivatives or acetonides of podophyllotoxin diol results in little or no activity of the compound.

Since inhibitor molecules are also relatively nonpolar, they can freely permeate cell membranes and IGF-1R receptors, but may also be sufficiently polar to be adequately soluble in water. The polarity of the molecule is determined by the number and nature of the oxygen functional groups. The polarity is assumed to be optimal when the solubility in water is between the solubility of deoxypodophyllotoxin (ie about 0.01 mM) and the solubility of grapephytotoxin (about 0.1 to 0.2 mM). Uncharged or highly polar groups should be present on this molecule.

The present invention relates to the use of a compound of formula (I) as an inhibitor of tyrosine phosphorylation of the insulin-like growth factor-1 receptor.

Wherein the distance between the carbon atom of the methylene group and the carbon atom of the methoxy group is 0.85 to 1.05 nm;

R is OH, OCH ₃ , OC ₂ H ₅ or C _1-5 linear or branched hydrocarbon chain, optionally having 1 to 3 oxygen functional groups, and optionally carbon (5) in the upper benzene ring Also forms bonds;

R ₁ may be the same or different, which is OH or OCH ₃ ,

n is 0-2.

Oxygen functional group as used herein means a hydroxy, oxo, carboxy, methoxy, methylenedioxy, lactone, ether and / or ester group.

One group of compounds that can be used in the present invention have the following formula (Ib).

Wherein R is OH, OCH ₃ , OC ₂ H ₅ , CH ₃ , C ₂ H ₅ or C ₂ H ₄ OH;

R ₁ and n are as defined above.

Substituents R of the compounds belonging to this group may be in the R or S position relative to the lower benzene ring.

Compounds of formula I can be prepared by the following representative synthesis methods:

Examples of compounds that can be prepared in this manner include:

6- (3,4,5-trimethoxy-alpha-hydroxy-benzyl) -1,3-benzodioxole (IA),

6- (3,4,5-trimethoxy-alpha-methoxy-benzyl) -1,3-benzodioxole (Ib),

6- (3,4,5-Trimethoxy-alpha-methyl-benzyl) -1,3-benzodioxol (Ic).

Another group of compounds that can be used by the present invention have the following formula (II).

Wherein R ₂ , R ₃ and R ₄ may be the same or different, which is H, OH, OCH ₃ , OC ₂ H ₅ , or both R ₂ and R ₃ are methylenedioxy groups, or R ₃ And R ₄ are all acetonide, carbonate or methylenedioxy groups;

R ₁ and n are as defined above.

When the substituents R ₂ , R ₃ and R ₄ are not represented as oxo groups, these substituents may be in the alpha- or beta-position. The lower benzene ring is preferably in the alpha-position.

Compounds of formula (II) can be prepared by the following representative synthesis methods:

In this scheme, NBS is N-bromosuccinimide, PPA is polyphosphoric acid, TTN is thallium nitrate trihydrate, p-TSA is p-toluenesulfonic acid, DMF is N, N-dimethylformamide , LDA is lithium dialkylamide.

Examples of compounds that can be prepared in this manner include:

1- (4-methoxy-phenyl) -6,7-methylenedioxy-1,2,3,4-tetrahydronaphthalene (IIa),

3-hydroxy-1- (4-methoxy-phenyl) -6,7-methylenedioxy-1,2,3,4-tetrahydronaphthalene (IIb),

1- (3,4,5-trimethoxy-phenyl) -6,7-methylenedioxy-1,2,3,4-tetrahydronaphthalene (IIg),

3-hydroxy-1- (3,4,5-trimethoxy-phenyl) -6,7-methylenedioxy-1,2,3,4-tetrahydronaphthalene (IIh).

Compounds of formula (II) may also be prepared by the following synthesis methods:

Examples of compounds that can be prepared by this method include the following:

2-hydroxy-1- (4-methoxy-phenyl) -6,7-methylenedioxy-1,2,3,4-tetrahydronaphthalene (IIc),

1- (4-methoxy-phenyl) -2-oxo-6,7-methylenedioxy-1,2,3,4-tetrahydronaphthalene (IId),

2-hydroxy-1- (3,4,5-trimethoxy-phenyl) -6,7-methylenedioxy-1,2,3,4-tetrahydronaphthalene (IIi),

1- (3,4,5-trimethoxy-phenyl) -2-oxo-6,7-methylenedioxy-1,2,3,4-tetrahydronaphthalene (IIj).

Compounds of formula II may also be prepared as follows:

Examples of compounds that can be prepared in this manner include:

2,3-dihydroxy-1- (4-methoxy-phenyl) -6,7-methylenedioxy-1,2,3,4-tetrahydronaphthalene (IIe),

1- (4-methoxy-phenyl) -2,3-methylenedioxy-6,7-methylenedioxy-1,2,3,4-tetrahydronaphthalene (IIf),

2,3-dihydroxy-1- (3,4,5-trimethoxy-phenyl) -6,7-methylenedioxy-1,2,3,4-tetrahydronaphthalene (IIk),

1- (3,4,5-trimethoxy-phenyl) -2,3-methylenedioxy-6,7-methylenedioxy-1,2,3,4-tetrahydronaphthalene (IIl).

A detailed description of the reaction conditions in this synthesis is described in Advanced Organic Chemistry, Jerry March (ed.), 4th edition, Wiley-Interscience Publication, New York, 1992.

Another group of compounds that may be used by the present invention are compounds of formula III.

Wherein R ₂ , R ₅ , R ₆ may be the same or different, which is H, OH, OOCH ₃ , OOCH ₂ CH ₃ , OCH ₃ or OC ₂ H ₅ , or both R ₅ and R ₆ are ether Or lactones;

R ₁ and n are as defined above.

When substituent R ₂ is a free hydroxy group, this substituent R ₂ cannot be in the alpha position like podophyllotoxin. Other R ₂ substituents, except oxo groups, may be in the alpha- or beta-position. Note that the lower benzene ring is in the alpha position, with beta bonds between carbons 8 and 9 and alpha bonds between carbons 8 'and 9', so deoxypodophyllotoxins and grapephyllo As in toxins, they form a trans configuration.

The present invention relates in particular to the use of any of the relatively nontoxic cyclolignans such as epipodophyllotoxin, deoxypodophyllotoxin and acetylpodophyllotoxin as an inhibitor of tyrosine autophosphorylation of the insulin growth factor-1 receptor For example, the use of more cytotoxic and tissue stimulatory compounds of podophyllotoxin and 4'-demethyl-podophyllotoxin is excluded.

Some compounds of formula III are naturally occurring in plants, for example deoxypodophyllotoxins and podophyllotoxins. In the manufacturing of the material in pure form, for example, the dried and pulverized rhizome of Modi (Podophyllum emodi) or grape pilryum pel tatyum (Podophillum peltatum) pilryum the grapes are extracted with an organic solvent. This extract is then filtered and concentrated on silica gel. Fractions containing this material are collected, and further purified by lysosomal staphylophyllium titanium by chromatography using acidic alumina, silica gel and the like, and finally recrystallized. Podophyllotoxin can be used as a starting material in the synthesis of its less toxic derivatives.

Epipodophyllotoxins are readily prepared from podophyllotoxins. 5 mg of grapephytotoxin are dissolved in 2.5 ml of acetone. 0.5 ml of concentrated HCl is added to the solution, and the mixture is boiled for 2 hours. The solution is then neutralized with aqueous NaHCO ₃ (about 0.5 g in 5 ml), then the acetone is evaporated and the product epipodophyllotoxin is extracted with ethyl acetate.

Acetylpodophyllotoxin (acetate derivative of podophyllotoxin) can be prepared from grapephytotoxin by incubating 0.1 mg of grapephytotoxin, 1 ml of acetic anhydride, and 1 ml of pyridine together at 50 ° C. for 16 hours. The reagents were then partially evaporated, 10 ml of water and 10 ml of ethyl acetate were added, then the product was extracted from the aqueous phase.

Acetonide and methylenedioxy derivatives can be prepared using diols obtained by reducing the lactone rings of naturally occurring lignans according to standard methods as starting materials.

Further examples of compounds of formula III include the following:

Podophyllotoxin and 4'-demethyl-deoxypodophyllotoxin.

The present invention also relates to novel compounds of formula (I)

Formula I

In the above formula,

R is OH, OCH ₃ , OC ₂ H ₅ , CH ₃ , C ₂ H ₅ or C ₂ H ₄ OH;

R ₁ and n are as defined above,

Provided that when R ₁ is OCH ₃ and n is 2, R is not OH,

When R is CH ₃ or C ₂ H ₅ , R ₁ is OH and n is 1 or 2,

If R is C ₂ H ₅ , R ₁ is not OH.

The molecules of the present invention must be relatively rigid in order to keep the distance between the two substituents within a predetermined range (0.95 ± 0.10 nm; 9.5 ± 1.0 ms). Since the ring structure of the hydrocarbon chain will inhibit the rotation or movement of the benzene ring, it will also inhibit lactone formation.

The present invention also relates to novel compounds of formula (II).

Formula II

Wherein R ₂ , R ₃ and R ₄ may be the same or different, which is H, OH, O, OCH ₃ , or both R ₂ and R ₃ are methylenedioxy groups, or R ₃ and R ₄ is all an acetonide, carbonate or methylenedioxy group;

R ₁ and n are as defined above.

In order to design inhibitors of IGF-1R tyrosine kinase for therapeutic purposes, it is very important not to crosslink the inhibitor with insulin receptor kinase which is highly homologous to the IGF-1R. Co-inhibitory action of the insulin receptor will elicit diabetic responses in vivo. This reaction causes very serious side effects, which can not be overcome by insulin treatment because receptor kinases are blocked. The inventors have found that grapephytotoxin and its derivatives are more potent IGF-1R inhibitors than tyropostin-based compounds and do not interfere with insulin receptor tyrosine kinase at all. These staphylotoxins and derivatives thereof do not interfere with tyrosine phosphorylation of epidermal growth factor, platelet derived growth factor or fibroblast growth factor receptors.

Grape filotoxin has been used for cancer treatment for a long time, but when it is administered to a patient it causes unacceptable side effects. Anticancer effects and side effects were due to inhibition of assembly of microtubules and blocking mitosis. Recently, it has been found that grapephytotoxin and some of its less toxic analogs are very potent and play an important role as survival factors for cancer cells as specific inhibitors of tyrosine phosphorylation of the insulin-like growth factor-1 receptor. To inactivate IGF-1R, a concentration 100 times lower than the concentration necessary for the antimicrotubule effect of grapephytotoxin was sufficient. Most importantly, grapephytotoxin and analogs thereof do not inhibit the insulin receptor (a receptor very homologous to IGF-1R). In addition, the grape filotoxin and its analogs inhibit none of the other major growth factor receptor kinases.

Relatively non-toxic compounds of formula (I) can be used for the treatment of IGF-1R dependent diseases such as cancer, arteriosclerosis, including preventing coronary artery restenosis, psoriasis and acromegaly after vascular surgery.

A pharmaceutical composition comprising a compound of formula (I) and a physiologically acceptable carrier and, if desired, an adjuvant may be administered to a patient by any route of administration, eg, parenteral, preferably by intravenous infusion or topical, such as by patch. May be administered.

The present invention relates to the use of a novel compound of formula (I) or formula (II) as a medicament, specifically in the manufacture of a medicament for the treatment of cancer.

The biological experiments suggested that sub-micromolar concentrations of fiphytotoxin or a less toxic analog such as deoxypodophyllotoxin or epipodophyllotoxin may be sufficient to cause tumor cell death. However, it is believed that it is important that the plasma concentration of the inhibitor is kept constant for a long time so that the inhibitor can saturate all IGF-1R continuously and in this way permanently kill as many malignant cells as possible. Thus, successive injections of the piphylotoxin derivatives while monitoring plasma concentrations can be a therapeutic alternative to repeated (eg daily) infusions, which may result in a repeated response of IGF-1R between treatments. have.

As a result, the present invention also provides a method of administering a pharmaceutical composition containing a compound of Formula I and a physiologically acceptable carrier to a tumor patient by a constant infusion method for a time sufficient to delay tumor progression or to eliminate the tumor, the plasma of the compound Adjusting the infusion rate and adjusting the infusion rate to adjust the plasma level to between 0.05 and 5.0 μM (depending on the general toxicity of the compound).

In the case of tumors that are not completely dependent on IGF-1R, the compounds of the present invention may be useful for sensitizing tumor cells to the effects of other anticancer drugs.

material

chemical substance

Cell culture reagents, i.e., fetal bovine serum and antibiotics, were purchased from Gibco, Sweden. All other chemicals used were purchased from Sigma (St. Louis, Missouri, USA) unless otherwise noted. Mouse monoclonal antibodies against phosphotyrosine (PY99) and polyclonal antibodies against the α-subunit of IGF-1R (N20) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Monoclonal antibodies against the α-subunit of IGF-1R (IR-3) were purchased from Oncogene Science (New York, USA). Deoxypodophyllotoxins and podophyllotoxins (purity 99.97%) and acetylpodophyllotoxins, podophyllotoxins and 4'-demethylpodophyllotoxins (purity> 95%) were donated from Analytecon SA, Switzerland.

Cell culture

Human malignant melanoma cell lines SK-MEL-2, SK-MEL-5 and S-MEL-28, prostate carcinogenic cell line PC-3 and breast cancer cell line MCF-7 were obtained from the American Tissue Culture Collection. Malignant melanoma cell lines BE and FM55 were obtained from Professor R. Kiessling (CCK, Karolinska Hospital, Stockholm, Sweden). R- and P6 cell lines were donated by Professor R. Baserga (Thomas Jefferson University, Philadelphia, Pennsylvania, USA). All cell lines were cultured in minimal essential medium containing 10% fetal bovine serum, glutamine, 1% benzylpenicillin and streptomycin. The cells were grown in a monolayer of tissue culture flasks in a humidified incubator maintained at 37 ° C. in 95% air / 5% CO ₂ atmosphere. For this experiment, cells were cultured in either 35 mm or 60 mm plastic dishes or 96 well plastic plates. The experiment was initiated under subconfluent growth conditions.

Human chronic myeloma leukemia K562 / S sub-K562 / Vcr30 cell lines and acute myeloma leukocyte cell lines HL60 / O and HL60 / Nov were obtained from ATCC. The K562 / S and HL60 / O were wild type (non-resistant) cells, while the K562 / Vcr30 and HL60 / Nov were cytostatic cell lines. All leukemia cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 2 mM glutamine, 1% benzyl-penicillin and streptomycin. These cells were grown in tissue culture flasks in a humidified incubator maintained at 37 ° C. under 95% air / 5% CO ₂ atmosphere. For this experiment, 25,000 cells were cultured in 60 mm plastic dishes or 96-well plastic plates. Experiments on leukemia cells were performed in collaboration with fellow professor Sigurd Vitols (class of pharmacology at Karolinska Hospital, Stockholm, Sweden).

Way

Cell Growth and Survival Assay

Cell Proliferation Kit II (Roche Inc.) is based on the colorimetric change of the yellow tetrazolium salt XXT in the orange formazan dye by the respiratory process of viable cells [Roehm, NW et al., J. Immunol. Methods 142: 257-265, 1991. Cells seeded at a concentration of 5000 / well in 100 μl medium in a 96-well plate were treated with a given concentration of different drug. Cells were treated 24 hours or 48 hours after incubation with the XXT labeling mixture, according to the manufacturer's protocol. After 4 hours, the formazan dye was quantified using a scanning multiwell spectrometer equipped with a 495 nm filter. Survival cell number and absorbance were directly correlated. Untreated cells seeded at a concentration of 1000-10,000 cells / well were used to draw increasingly standard absorbance curves at a rate of 1000 cells / well. All standards and experiments were performed in triplicate.

Immunoprecipitation and Determination of Protein Content

Isolated cells were lysed in 10 ml of ice cold PBSTDS containing protease inhibitors (Carlsberg, M., et al., J. Biol. Chem. 271: 17453-17462, 1996). 50 μl Protein A or G agarose was added to 1 ml sample and it was incubated for 15 minutes at 4 ° C. in an orbital shaker. After centrifugation at 10,000 r / min for 10 minutes at 4 ° C, the supernatant was taken. Protein content was determined by dye binding assays using reagents purchased from Bio-Rad. Bovine serum albumin was used as a standard. 15 μl of Protein G plus agarose and 5 μl of anti-IGF-1R were added. After incubation in an orbital shaker at 4 ° C. for 3 hours, the precipitate was collected by pulse centrifugation in a microcentrifuge (14,000 × g) for 10 seconds. The supernatant was discarded and the pellet washed three times with PBSTDS.

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Protein samples were dissolved in 2 × sample buffer containing Laemli buffer and 0.5% methanol and heated at 96 ° C. for 5 minutes. Samples were separated by SDS-PAGE using 4% stacking gels and 7.5% separation gels. In all experiments molecular weight markers (Bio Rad, Sweden) were developed simultaneously.

Western Blot Method

After SDS-PAGE, the protein was transferred to nitrocellulose membrane (Hybond, Amersham, UK) overnight and then blocked in 4% skim milk powder in PBS and 0.02% Tween 20 solution (pH7.5) at room temperature for 1 hour. . Incubated for 1 hour at room temperature with the primary antibody, then washed three times with PBS containing Tween and then incubated with secondary antibody for 1 hour at room temperature. After three more washes, the membranes were incubated with streptavidin-labeled horseradish peroxidase for 30 minutes and detected using the Amersham ECL system (Amersham, UK). The film was scanned with Fluor-S (BioRad).

In Vitro Assay of IGF-1R Autophosphorylation

IGF-1R tyrosine autophosphorylation was analyzed by sandwich ELISA assay. In summary, 96-well plates (Immunolon, Nunc.) Were coated with 1 μg / well of monoclonal antibody Ab-5 (LabVision) (an antibody to the IGF-1R beta subunit) overnight at 4 ° C. This plate was blocked with 1% BSA in PBS Tween for 1 hour, after which total protein lysate from P6 cell line was added at a concentration of 80 g / well. Total protein lysate from the R-cell line was used as a negative control. The observed compound was added to tyrosine kinase buffer containing no ATP for 30 minutes at room temperature before kinase was activated with ATP. Kinase assays were performed using the Sigma kit. After the spectrometer measurement, the IC50 value of the inhibitor was measured using the regression function of the Statistica program.

Experiment 1.Effect of grapephytotoxin and other biologically active phenolic compounds on phosphorylation of IGF-1R in cultured melanoma cells

Melanoma cells (cell line FM55) were seeded in 6 cm dishes, that is, supplemented with minimal essential medium (10% calf fetal serum (FCS)) at a concentration of 10,000 cells / cm 2. When the cell concentration reached 65,000 cells / cm 2, they were treated with genistein, tamoxifen, quercetin and grapephytotoxin at 0, 1, 15 or 60 μM in final concentration culture medium for 1 hour. Treatment with 0 μM represents an untreated control. The cells were then separated and immunoprecipitated with IGF-1R. The immunoprecipitates containing purified IGF-1R were fractionated by gel electrophoresis. Phosphorylation of IGF-1R was confirmed with anti-phosphotyrosine antibodies using Western blot. The signal obtained indicates phosphorylated IGF-1R and the intensity of the signal indicates the amount of phosphorylated IGF-1R. Detailed description of the method used is described above. Intensity was quantified by a scanner measuring the optical density (OD) of the signal. For control cells, the OD was set at 100%. Blank (OD 0%) represents the background. The results shown in Table 1 below represent the mean values for three experiments.

IGF-1R Phosphorylation Level in Intact State Cells compound 1 μM 15 μM 60 μM Genistein 100 96 35 Tamoxifen 95 20 10 Quercetin 100 105 96 Grape phytotoxin 8 4 2

These results showed that grapephytotoxin almost completely inhibited IGF-1R phosphorylation at three concentrations, whereas only genistein showed a partial inhibitory effect at 60 μM and quercetin had no effect at all. Able to know.

Experiment 2. Effect of Staphylococtotoxin Derivative on IGF-1R Autophosphorylation in Cultured Melanoma Cells

FM55 melanoma cells were cultured in the same manner as described in Experiment 1. When the dish reached a density of 65,000 cells / cm 2, the dish was treated with 0.05 μM of grapephytotoxin, deoxypodophyllotoxin, acetylpodophyllotoxin, epipodophyllotoxin, 4′-demethyl-podophyllo for 1 hour. Treatment with toxin and grapephytotoxone. The cells were then collected for analysis as described above and quantified IGF-1R autophosphorylation. The values shown in Table 2 below represent the average of three experiments.

Inhibitory Effect on IGF-1R Autophosphorylation in Intact Cells Related to Staphylococtoxin compound Relative efficacy Grape phytotoxin One Deoxypodophyllotoxin 0.8 Acetylpodophyllotoxin 1.3 Epipodophyllotoxins 0.5 4'-demethylpodophyllotoxin 0.5 Grape phytotoxin 0.3

The results show that acetylpodophyllotoxin, grapephylotoxin and deoxypodophyllotoxin are potent inhibitors of IGF-1R phosphorylation.

Experiment 3. Effect of Dose Response on Staphylococtotoxin and Deoxypodophyllotoxin on Survival Rate of Solid Tumor Cells.

Five different types of cell lines were seeded in 96-well plates (100 μl of medium volume in wells) at a concentration of 10,000 cells / cm 2 in minimum essential medium (supplemented with calf fetal serum). When the cell concentration reached a concentration of 65,000 cells / cm 3, they were treated for 48 hours with different doses of grapephytotoxin and deoxypodophyllotoxin. Cell viability was then analyzed (see above). From this, it was found that the cell viability was reduced by 50% through IC 50 values (calculated as concentration) for each inhibitor and cell line. The results are based on four different experimental results.

IC50 (μM) for cell viability Cell line origin Grape phytotoxin Deoxypodophyllotoxin SK-MEL-28 Melanoma 0.05 0.04 BE Melanoma 0.05 n.d. FM55 Melanoma 0.04 0.04 MCF-7 Breast cancer 0.07 0.03 PC-3 Prostate cancer 0.06 n.d. n.d. : Not measured

These results showed that both grape filotoxin and deoxypodophyllotoxin are useful inhibitors for tumor cell survival.

Experiment 4. Dose Response Effects on Different Staphylotoxin Analogs

FM55 melanoma cells were cultured in the same manner as described in Experiment 3, and the cells were treated with different doses of staphylotoxin analogs as described in Experiment 3 above. The results (IC 50 values) are shown in Table 4 below.

IC50 (μM) for survival of FM55 cells compound IC50 Grape phytotoxin 0.05 Deoxypodophyllotoxin 0.04 Acetylpodophyllotoxin 0.03 4'-demethylpodophyllotoxin 0.04

The results showed that all of the analogs tested were useful.

Experiment 5. Effect of Dose Response on Leukemia Cells of Staphylotoxin Analogues

The leukemia cell lines K562 / S, K562 / Vcr 30, HL60 / 0 and HL60 / Nov were found to express IGF-1R. This was analyzed by Western blot analysis as described in the methods described above and in Experiments 1 and 2. The four leukemia cell lines were seeded in 96-well plates (medium volume = 100 μl in wells) in RPMI40 medium supplemented with calf fetal serum. After 24 hours, grapephytotoxin, deoxypodophyllotoxin and other derivatives were added at different concentrations for 72 hours. Cell viability was then analyzed (see above). IC50 values for each inhibitor and cell line are shown below (Table 5). The results are based on three different experiments.

IC50 (nM) for survival of leukemia cell lines K562 / S K562 / Vcr30 HL60 HL60 / Nov Grape phytotoxin 4 7 3 2 Deoxypodophyllotoxin 3 4 3 2 Acetylpodophyllotoxin 80 140 48 46 Epipodophyllotoxins 195 450 127 90 Grape phytotoxone > 500 > 500 > 500 > 500 4'-demethyl-podophyllotoxin 22 50 20 18

The results show that deoxypodophyllotoxin has a very potent cytotoxic effect on human leukemia cells unexpectedly. This effect cannot be explained solely by its action on IGF-1R. The effects of acetylpodophyllotoxin and epipodophyllotoxin were as expected for the IGF-1R inhibitors.

conclusion

Certain cyclolignans such as podophyllotoxin and several analogs, deoxypodophyllotoxins, have been analyzed in their original cells and have been shown to be very specific and useful inhibitors of IGF-1R tyrosine kinase. When administered to cells intact, EC50 was 0.02 to 0.06 μM.

Staphylotoxin-induced inactivation of the insulin-like growth factor-1 receptor caused widespread cell death in malignant cells, whereas cells without the insulin-like growth factor-1 receptor were resistant. The non-toxic derivative pyropodophylline was equally effective in inhibiting grapephytotoxin and insulin-like growth factor-1 receptor activity and inducing cell death. Such novel mechanisms of podophyllotoxin and derivatives thereof may be useful for the treatment of cancer and other IGF-1R dependent diseases.

Claims (14)

Use of compounds of formula I as inhibitors of tyrosine phosphorylation of insulin-like growth factor-1 receptors: Formula I In the above formula, The distance between the carbon atom of the methylene group and the carbon atom of the methoxy group is 0.85 to 1.05 nm; R is OH, OCH ₃ , OC ₂ H ₅ or C _1-5 linear or branched hydrocarbon chain, optionally with double bonds, optionally with 1-3 oxygen functional groups, optionally benzene May form a bond with a carbon atom in ring A; R ₁ may be the same or different, which is OH or OCH ₃ and n is 0-2. Use according to claim 1, wherein the compound of formula I is as follows: Formula I In the above formula, R is OH, OCH ₃ , OC ₂ H ₅ , CH ₃ , C ₂ H ₅ or C ₂ H ₄ OH; R ₁ and n are as defined in claim 1. The use according to claim 1, wherein the compound has formula II: Formula II Food, R ₂ , R ₃ and R ₄ may be the same or different, which may be H, OH, OCH ₃ , or both R ₂ and R ₃ are methylenedioxy groups, or R ₃ and R ₄ are all acetonides, Carbonate or methylenedioxy group; R ₁ and n are as defined in claim 1. Use according to claim 1, wherein the compound has the formula Formula III Food, R ₂ , R ₅ and R ₆ may be the same or different, which is H, OH, OOCH ₃ , OOCH ₂ CH ₃ , OCH ₃ or OC ₂ H ₅ , or both R ₅ and R ₆ are ether or lactone And; R ₁ and n are as defined in claim 1, Provided that when R ₂ is OH, this substituent may not be at the alpha position. The use according to claim 4, wherein the compound is selected from the group consisting of epipodophyllotoxin, deoxypodophyllotoxin and acetylpodophyllotoxin. Compound of formula (I) Formula I Wherein R is OH, OCH ₃ , OC ₂ H ₅ , CH ₃ , C ₂ H ₅ or C ₂ H ₄ OH; R ₁ and n are as defined in claim 1, Provided that when R ₁ is OCH ₃ and n is 2, R is not OH. A compound of formula II: Formula II Food, R ₁ , R ₃ and R ₄ may be the same or different and may be H, OH, O, OCH ₃ ; Or R ₂ and R ₃ are both methylenedioxy groups, or R ₃ and R ₄ are all acetonide, carbonate or methylenedioxy groups; R ₁ and n are as defined above. A compound of formula (I), formula (II) or formula (III) used as a medicament. Use of a compound of Formula (I), (II) or (III) in the treatment of IGF-1R dependent diseases such as cancer, arteriosclerosis, psoriasis and acromegaly. Use of a compound according to claim 6 or 7 in the manufacture of a medicament for the treatment of cancer. Use of a compound of formula III in the manufacture of a medicament for the prevention or treatment of cancer: Formula III Food, R ₂ , R ₅ and R ₆ may be the same or different, which is H, OH, OOCH ₃ , OOCH ₂ CH ₃ , OCH ₃ or OC ₂ H ₅ , or both R ₅ and R ₆ are ether or lactone And; R ₁ and n are as defined in claim 1, Provided that when R ₂ is OH, this substituent may not be at the alpha position. Use of a compound in the prevention or treatment of leukemia according to claim 11, wherein the compound is selected from the group consisting of epipodophyllotoxin, deoxypodophyllotoxin and acetylpodophyllotoxin. A pharmaceutical composition comprising a compound of Formula I, Formula II or Formula III and a physiologically acceptable carrier. Administering to the tumor patient by constant infusion methods a pharmaceutical composition containing a compound of formula (I), a compound of formula (II), or a compound of formula (III) and a physiologically acceptable carrier, for a time sufficient to delay tumor progression or eliminate the tumor, Monitoring the plasma level of the compound and adjusting the infusion rate to maintain the plasma level between 0.05 and 5.0 μΜ.