WO2007051622A1

WO2007051622A1 - Lead structures for cytostatic compounds based on spiro compounds

Info

Publication number: WO2007051622A1
Application number: PCT/EP2006/010523
Authority: WO
Inventors: Michael Lalk; Peter Langer; Gopal Bose; Patrick J. Bednarski; Karin Bracht
Original assignee: Ernst-Moritz-Arndt-Universität Greifswald
Priority date: 2005-11-03
Filing date: 2006-11-02
Publication date: 2007-05-10
Also published as: DE102005052501A1

Abstract

The invention relates to lead structures of general formula X for the treatment of tumor diseases. The invention further relates to pharmaceutical compositions containing said compounds as well as the use of said compounds for treating diseases.

Description

GUIDANCE STRUCTURES FOR CYTOSTATIC COMPOUNDS BASED ON SPIRO COMPOUNDS

The present invention relates to novel biologically active compounds for the treatment of diseases, more particularly, the present invention relates to novel lead structures for cytostatic compounds for the therapy of tumor diseases. The invention further relates to pharmaceutical compositions containing these compounds and the use of these compounds in the treatment of diseases.

Background of the invention

A tumor (tumor, neoplasia) consists of the body's own cells, which multiply independently of the actual "blueprint of the body." A malignant tumor (cancer) occurs when the tumor tissue destructively penetrates into the healthy neighboring tissue and swept away tumor cells in other organs secondary tumors ( A cure requires the elimination of all malignant cells (curative therapy) .If this is not possible, attempts can be made to slow their growth in order to prolong the life of the patient (palliative therapy), however, the drug therapy is available the difficulty that the malignant cells are endogenous and have little specific metabolic properties.

Cytotoxic agents are cytotoxic substances that are particularly targeted to cells that target cell division (mitosis). Rapidly dividing, malignant cells are preferentially damaged. Damage to cell division processes not only slows down the proliferation, but can also trigger the phenomenon of apoptosis (self-destruction of the affected cells). Low cell division tissue remains largely unaffected. This also applies to malignant tumors from differentiated, rarely dividing cells. However, some healthy tissues have a high rate of mitosis physiologically and cytostatic therapy inevitably affects these tissues.

Thus, cytostatic compounds play an important role in the treatment of cancer and thereby form an important group among the pharmaceutically relevant organic compounds. The treatment of cancer is an urgent goal of medicine, and in addition to the surgical removal of tumors chemotherapy is the possibility the treatment of a variety of non-solid tumors or the treatment of metastasizing tumors [a) Zips, Daniel; Thames, Howard D .; Baumann, Michael. New anticancer agents: in vitro and in vivo evaluation. In Vivo (2005), 19 (1, Spec. Iss.), 1-7; b) Kim, Ryungsa. Recent advances in understanding the cell death pathways activated by anticancer therapy. Cancer (New York, NY, United States) (2005), 103 (8), 1551-1560].

As with bacterial infections, resistance to single or a group of cytostatic drugs has also been reported more frequently in tumor cells [a) Filipits, Martin. Mechanisms of Cancer: multidrug resistance. Drug Discovery Today: Disease Mechanisms (2004), 1 (2), 229-234; b) Righetti, Sabina C; Gatti, Laura; Beretta, Giovanni L .; Zunino, Franco; Perego, Paola. Resistance to antitumor drugs in the post-genomic era. Recent Research Developments in Molecular Pharmacology (2002), 1, 167-184]. These resistances are due to the mutation of the genome of tumor cells and cause z. B. enzymes are formed that allow degradation of cytotoxic agents.

Cytostatic agents are established substances that find wide application in the treatment of tumors [H. D. Brun, cytostatics primer, Schattauer, Stuttgart, 1985]. Prominent representatives of this class of drugs are the compounds of the type of alkylating agents (eg chlorambucil, melphalan, thiotepa and busulfan), platinum compounds (eg cisplatin, carboplatin), topoisomerase inhibitors (eg camptothecin, etoposide, doxorubicin), mitotic poisons (eg taxol, vinblastine ) and antimetabolites (eg 5-fluorouracil, methotrexate, hydroxyurea). These compounds can be classified according to their mechanism of action in compounds that:

- have alkylating properties (alkylating agents)

- Intercalation compounds with nucleic acids (Pt compounds) inhibit the formation of microtubules or prevent their degradation (mitosis toxins)

- inhibit nucleic acid synthesis (antimetabolites).

There is already resistance in the tumor cells to a number of this compound. In order to counteract the formation of new resistance, a combination approach is often used in therapy. In addition to the actual cytostatic compounds, active ingredients are administered which counteract a mutation of the genome towards resistance. Cytotoxic natural products are often used as lead structures for the development of new cancer therapeutics [Kim, J .; Park, EJ Curr. Med. Chem. Anti-Cancer Agents, 2002, 2, 485]. Often toxic fungi contain natural products with cytotoxic properties, such as the "jack-o-latern" fungus Omphalotus illudens In 1950, Illudin S and M were isolated as the cytotoxic components of O. illudens [a) Saever, FJJNY Bot. Garden 1938, 263; b) Seaver, FJJNY Bot. Garden 1939, 236; c) Nakanishi, K., Tada, M., Yamada, Y., Ohashi, M., Komatsu, N., Terekawa, H. Nature 1963, 197, 292] After elucidating their structure, [a) Anchel, M., Hervey, A., Robbins, WJ Proc., Nat., Acad., Sci., USA, 1950, 36, 300. b) McMorris, T. C. Anchel, MJ Am. Chem. Soc. 1963, 85, 831; c) McMorris, T.C., Anchel, MJ Am. Chem. Soc., 1965, 87, 1594] have isolated many related structures from O. illudens and other fungi [Ayer, WA; Browne, LM Tetrahedron 1981, 37, 2199]. All compounds show a cytotoxic effect, with Illudin S and M showing very strong activity and the dehydroderiavte and aeylated compounds showing poor activity [McMorris, T.C., None, MJ; Wang, W., Estes, L A; Montoya, MA; Taetle, RJ Org. Chem. 1992, 57, 6876]. Due to its cytotoxic activity, the synthesis of illudin analogs is of particular pharmacological interest [Padwa, A .; Curtis, EA; Sandanayaka, VPJ Org. Chem. 1996, 61, 73].

Object of the invention

The foreseeable after the prior art training of resistance to one or more classes of substances would make the treatment of tumor diseases difficult or impossible. Therefore, the discovery of new cytostatic compounds is an urgent task in pharmaceutical research and new lead structures for cytostatic compounds are a promising approach for the treatment of tumor diseases.

It is therefore an object of the invention to provide novel lead structures for compounds which are suitable for use in the therapy of tumor diseases.

This object is achieved by compounds of the general formula X:

in which

R is selected from: (Ci-Ci _O) -alkyl, wherein the alkyl may be straight or branched, (C ₁ - C ₀₎ alkoxy, wherein the alkoxy may be straight or branched;

R ^{1 is} selected from: (C 1 -C 10) -alkyl, wherein the alkyl may be straight, branched or cyclic;

R ² is selected from: (C ₁ -C] ₀₎ - alkyl, where the alkyl is straight, branched, may be cyclic or bicyclic, (C] -C _O) -alkenyl, wherein the alkenyl straight, branched, cyclic or bicyclic and combinations thereof, such as spiro rings and bicyclic ring systems;

R ³ is selected from: (Cj -C ₀₎ - alkyl, where the alkyl is straight, branched, may be cyclic or bicyclic, (Ci-C) ₀₎ - alkenyl, wherein the alkenyl straight, branched, may be cyclic or bicyclic and combinations thereof such as spiro rings and bicyclic ring systems;

R ⁴ is selected from: (C ₁ -C ₀₎ - alkyl, wherein the alkyl may be straight or branched; R ⁵ can either be unsubstituted or a hydroxy group; and their diastereomers, as well as the corresponding enantiomers and pharmaceutically acceptable salts and solvates of the compound, for the treatment of diseases.

In a preferred embodiment, the object is achieved by compounds according to formula Y:

in which

R is selected from: (C 1 -C 10) -alkyl, where the alkyl may be straight or branched,

C ₁₀ ) alkoxy, where the alkoxy may be straight or branched;

R ^{1 is} selected from: (C ₁ -C 10) -alkyl, where the alkyl may be straight or branched;

R ^{4 is} selected from:: (C 1 -C 10) -alkyl, wherein the alkyl may be straight or branched; and their diastereomers, as well as the corresponding enantiomers and pharmaceutically acceptable salts and solvates of the compound, for the treatment of diseases.

In a preferred embodiment, the object is achieved by a compound according to formula 1:

and their diastereomers, as well as the corresponding enantiomers and pharmaceutically acceptable salts and solvates of the compound, for the treatment of diseases.

In a further preferred embodiment, the object is achieved by a compound according to formula 4:

In another preferred embodiment, the object is achieved by a compound according to formula 5:

In a further preferred embodiment, the object is achieved by a compound according to formula 6:

In an additional preferred embodiment, the object is achieved by a compound according to formula 7:

In a preferred embodiment, the object is achieved by compounds according to formula Z:

in which

R ^{2 is} selected from: (C 1 -C ₁₀ ) -alkyl, where the alkyl may be straight, branched or cyclic, (C 1 -C ₁₀ ) -alkenyl, where the alkenyl may be straight, branched or cyclic; R ³ is selected from: _(! C -C ₀₎ - alkyl, where the alkyl is straight, branched or cyclic, _(Ci-C10) - alkenyl, wherein the alkenyl straight, branched or may be cyclic; R ⁵ can either be unsubstituted or a hydroxy group; and their diastereomers, as well as the corresponding enantiomers and pharmaceutically acceptable salts and solvates of the compound, for the treatment of diseases.

In a further preferred embodiment, the object is achieved by a compound according to formula 9:

In a further preferred embodiment, the object is achieved by a compound according to formula 10:

and their diastereomers, as well as the corresponding enantiomers and pharmaceutically acceptable salts and solvates of the compound, for the treatment of diseases. In a further preferred embodiment, the object is achieved by a compound according to formula 3:

In a further preferred embodiment, the object is achieved by a compound according to formula 2:

More recently, the syntheses of 1-hydroxyspiro [5.2] cyclooct-4-en-3-one derivatives [Böse, G .; Langer, P. Tetrahedron Lett. 2004, 3861], spiro [5.4] decenone derivatives and bicyclo [4.4.0] deca.l, 4-dien-3-one compounds [Böse, G .; Ullah, E .; Langer, P. Chem. Eur. J. 2004, 10, 6015]. However, these compounds are not dienes, i. they have one double bond less than the substances according to the invention.

Furthermore, Illudin S and M are described as having a very potent cytotoxic activity. However, this activity can not be deduced from the pharmaceutical activity of the specific substances of the invention, since the sole cytotoxicity is of no concern for the possibility of treatment of cancer and in particular leukemia and also says nothing about a cytostatic effect. Illudin S and M should therefore not be the subject of the present invention.

Likewise, the object is achieved by the use of a compound according to the present invention as a medicament, and by a pharmaceutical composition comprising a compound of the invention as mentioned above, together with suitable additives or excipients. The term "excipient" means according to the invention any non-toxic, solid or liquid filling, diluting or packaging material as long as it does not unduly adversely react with an inhibitor or the patient Liquid galenic adjuvants are, for example, sterile water, physiological saline, sugar solutions , Ethanol and / or oils Galenic adjuvants for the production of tablets and capsules may, for example, contain binders and fillers.

The compound is preferably present in the form of a depot substance or as a precursor together with a suitable, pharmaceutically acceptable dilution solution or carrier substance.

In one embodiment, the pharmaceutical composition contains other chemotherapeutic agents. These chemotherapeutic agents may include any of the chemotherapeutic agents commonly used in the art and well described in the art for cancer therapy (such as adriamycin (doxorubicin), BCNU (carmustine), busulfan, bleomycin, carboplatin, chlorambucil, cisplatin, dacarbazine (DTIC) , Docetaxel, epirubicin, etoposide (VP 16), 5-fluorouracil, gemcitabine, ifosfamide, interferon alpha, irinotecan (CPT 11), melphalan, methotrexate, mitomycin C, mitoxantrone, oxaliplatin, paclitaxel, prednimustine, procarbazine, ralitrexed, trofosfamide, vinblastine , Vincristine, vindesine, vinorelbine or others). The ultimate therapeutic will depend on the type of cancer and the personal needs of the patient.

The pharmaceutical composition according to the invention is preferably in the form of tablets, dragees, capsules, dripping solutions, suppositories, injection or infusion preparations for peroral, rectal or parenteral use. Such dosage forms and their preparation are known in the art.

The objects of the invention are achieved by the use of a compound according to the present invention together with suitable additives or excipients for the preparation of a medicament for the treatment of tumor diseases and preferably leukemia. Also, the objects of the invention are achieved by the use of a compound according to the present invention together with suitable adjuvants or excipients for the manufacture of a medicament for the treatment of leukemia.

In one embodiment, the compound is in the form of a depot substance or precursor, together with a suitable pharmaceutically acceptable diluent solution or vehicle.

A further preferred embodiment of the process of the present invention further comprises the step of chemically derivatizing the compounds as selected above. As used herein, in the context of the present invention a "derivative" is to be understood as meaning a compound derived from the compound according to the invention, which may be e.g. substituted by various residual groups, as well as mixtures of various of these compounds, e.g. can be processed on the basis of diagnostic data or data on the success or course of treatment matched to a "personalized" drug to the respective disease to be treated and / or the patient. In the context of the present invention, a "chemical derivatization" is to be understood as the process for a corresponding chemical change, e.g. the substitution of various residual groups. Preferably, chemical derivatization is performed for the purpose of achieving better bioavailability or reducing potential side effects.

In the context of the present invention, a "derivative" should also be understood to mean a precursor of a substance. In the context of the present invention, a "precursor" of a substance is to be understood as meaning a substance which is changed in the course of its administration for treatment by the conditions in the body (for example pH in the stomach or the like) or else by the substance Body is so metabolized after ingestion that form the active compound of the invention compound or derivatives thereof.

In one embodiment, the compound is employed in an amount in a drug sufficient to achieve the desired therapeutic effect. In a further embodiment of the present invention, the medicament that according to the is administered by various routes, for example, orally, parenterally, subcutaneously, intramuscularly, intravenously or intracerebrally. Appropriate doses may be readily obtained by one skilled in the art through routine experimentation and based on factors such as the concentration of the active ingredient, the body weight and age of the patient, and other patient or active ingredient related factors. The appropriate dose may be presented as a single dose or as divided doses, at appropriate intervals, for example as two, three, four or more sub-doses per day. The initial dosage in humans is accompanied by clinical monitoring of symptoms, usually the symptoms of the selected condition.

The invention will now be further elucidated hereinafter by way of example with reference to the accompanying figures, without, however, being limited to these examples. It shows

FIG. 1: Examples of the cytostatic spiro compounds.

FIG. 2: Graphical representation of the IC50 values of the compounds 1-10 shown in FIG.

Table 1 gives the IC50 values determined in the whole-cell assay for the compounds 1-10 shown in FIG. 1 as well as the comparative compounds.

Examples

The compounds shown in FIG. 1 were basically prepared by the method already known from the prior art in [G. Bad, P. Langer, Tetrahedron Lett. 2004, 3861-3863].

The cytostatic properties were assessed by a whole cell assay on the acute human leukemia cell line HL60. Rapid growth cells were exposed to the test compound for 48 hours. The IC50 values were determined from the MTT assay data (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolium bromide), which measures cell viability. It was found that the compounds 1 to 10 tested had an effect on the growth of this tumor cell line.

The results of the cell assay are summarized in Table 1 and FIG. In general, compounds with an inhibitory concentration of IC50 (inhibition of the growth of cells by 50%) of less than 20 μM is a promising candidate for the development of a cytostatic agent.

For the determination of the cytotoxic properties of the compounds shown in FIG. 1, according to Collins [Collins, Gallo, Gallagher "Continuous growth and differentiation of human myeloid leukaemic cells in suspension culture" Nature, 1977, 270: 347-349] and Gallagher [Gallagher, Collins, Trujillo, McCredie, Ahaern, Tsai, Metzgar, Aulakh, Ting, Ruscetti, Gallo "Characterization of the continuous, differentianting myeloid cell line (HL60) from a patient with acute promyelocytic leukemia blood, 1979, 54 (3):" The MTT test was carried out according to the instructions of Mosmann [Mosmann "Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays" J. Immun. Meth., 1983, 65: 55-63].

The 1-hydroxyspiro [2.5] cyclooct-4-enes 1, 4, 5, and 7 show antiproliferative properties comparable to the efficacy of carboplatin and 6-fluorouracil and stronger than that of busulfan. The effectiveness of compound 6 is in the same range as that of busulfan. In contrast, spiro [5.4] decenones 9, 10, 3 and cyclopropane 5 show only weak activity.

These results indicate that the presence of the 1-hydroxyspiro [2.5] cyclooct-4-ene group is indispensable for the cytostatic activity of the compounds.

The comparison of the obtained data shows very impressively that the developed compounds have a high potential to be used as cytostatic derivatives. In particular, the compounds 1, 4, 5 and 7 reach with their IC50 values the range that may be interesting for further development as a guide structure. IC50 values are found which reach or far exceed the inhibitory concentrations of established cytostatics. materials

All chemicals were obtained from SIGMA (Deisenhofen) and used without further purification.

Cell material (HLόO cells):

• Source: DSMZ (Braunschweig, No. ACC3)

Human leukemia line (type: acute myeloid leukemia, FAB subtype M2)

• Medium: RPMI 1640 + 10% FCS (with 15mg penicillin G and 27mg gentamicin fat / 500ml)

• Incubation: 37 ° C, 5% CO ₂ , 95% humidity

• round cells, single cell suspension

• Doubling time: approx. 24h

• Approx. 12 μm cell diameter

Carrying out the MTT assay:

The MTT assay was carried out according to the instructions of Mosmann [Mosman "Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays" J. Immun. Meth., 1983, 65: 55-63] with 2.5 mg MTT per liter of PBS (including 0.15M NaCl, 0.02M Na ₃ PO ₄ ), 20μl (freshly prepared each) were added per well and incubated for ₄ hours in an incubator (37 ° C, 5% CO ₂ ) Formazan crystals are incubated in The absorption was measured at 570 nm (ANTHOS 2010 plate photometer) MTT is taken up by metabolically active cells (endocytosis) and reduced to the corresponding formazan under NADH ⁺ consumption, which accumulates in Endosomes and lysosomes and is transported out of the cells via exocytosis.

Carrying out the cytotoxicity test:

• 5,000 cells / 50μl / well (= 100,000 cells / ml)

• zero control = 100 μl medium (1 row / plate)

• Control = 50 μl cells + 50 μl medium incl. Solvent DMSO (0.1%) (1 row per plate)

Compound = 50 μl cells + 50 μl compound dilution in medium (2 x 5 rows per plate, ie two compounds on one plate) In each case 5x1: 10 dilutions of the compounds are tested; the dilutions are made in the solvent (DMSO), the dilution to the final concentration in the well is then 1: 1000 in medium. The t _o plates (6 rows 100 μl medium / well, 6 rows 50 μl cells + 50 μl medium / well) are examined at time 0 of the treatment by MTT assay. The treated cells / plates are incubated for 48 h in the incubator (37 ⁰ C, 5% CO ₂ ) and then evaluated by MTT assay. The cells are treated immediately after sowing, there is no preincubation. The data of the photometer are transferred to Excel data sheets and evaluated: for this purpose, the T / Ck _o rr (%) values are plotted against the corresponding active substance concentration and the concentration corresponding to 50% growth inhibition (IC ₅₀ ) from the dose - Action curves determined as follows:

48h - OD _un treated, tö) / (OD _un b _e acts, 48h - OD _un treated, tθ) * 100

The IC ₅₀ values were determined as averages of several independent experiments (at least 3).

Table 1: IC50 values determined in the whole-cell assay for the test compounds and comparative compounds

Claims

claims

1. Compound of the general formula X:

in which

R is selected from: (C 1 -C 10) -alkyl, where the alkyl may be straight or branched, (C 1 -C ₁₀ ) -alkoxy, where the alkoxy may be straight or branched; R ¹ is selected from: (Ci-Cio) -alkyl, where the alkyl is straight, branched or may be cyclic;

R ² is selected from: (C ₁ -C ₀₎ - alkyl, where the alkyl is straight, branched, may be cyclic or bicyclic, (Ci -C ₀₎ - alkenyl, wherein the alkenyl straight, branched, may be cyclic or bicyclic and combinations thereof such as spiro rings and bicyclic ring systems;

R ³ is selected from: (C ₁ -C ₁₀₎ - alkyl, where the alkyl is straight, branched, may be cyclic or bicyclic, (Ci -C ₀₎ - alkenyl, wherein the alkenyl straight, branched, may be cyclic or bicyclic and combinations thereof such as spiro rings and bicyclic ring systems;

R ^{4 is} selected from:: (C 1 -C 10) -alkyl, wherein the alkyl may be straight or branched;

R ⁵ can either be unsubstituted or a hydroxy group; and their diastereomers, as well as the corresponding enantiomers and pharmaceutically acceptable salts and solvates of the compound, for the treatment of diseases.

2. A compound according to claim 1 having the formula Y:

in which R is selected from: (Ci-Cio) alkyl, wherein the alkyl may be straight or branched, (C ₁ -C _O) -alkoxy, wherein the alkoxy may be straight or branched;

R ^{4 is} selected from:: (C ₁ -C 10) -alkyl, wherein the alkyl may be straight or branched; and their diastereomers, as well as the corresponding enantiomers and pharmaceutically acceptable salts and solvates of the compound, for the treatment of diseases.

3. A compound according to claim 2 having the formula 1:

4. A compound according to claim 2 having the formula 4:

5. A compound according to claim 2 having the formula 5:

6. A compound according to claim 2 having the formula 6:

7. A compound according to claim 2 having the formula 7:

8. A compound according to claim 1 having the formula Z:

in which

R ^{2 is} selected from: (C 1 -C 10) -alkyl, where the alkyl may be straight, branched or cyclic, (C 1 -C 10) -alkenyl, where the alkenyl may be straight, branched or cyclic;

R ^{3 is} selected from: (C 1 -C ₁₀ ) -alkyl, wherein the alkyl may be straight, branched or cyclic, (C 1 -C ₁₀ ) -alkenyl, where the alkenyl may be straight, branched or cyclic;

9. A compound according to claim 8 having the formula 9:

10. A compound according to claim 8 having the formula 10:

11. A compound according to claim 8 having the formula 3:

12. A compound according to claim 1 having the formula 2:

A pharmaceutical composition comprising a compound according to any one of claims 1 to 12, together with suitable additives or excipients.

14. A pharmaceutical composition according to claim 13, characterized in that the compound is in the form of a depot substance or as a precursor together with a suitable, pharmaceutically acceptable diluting solution or carrier substance.

15. A pharmaceutical composition according to claim 13 or 14, characterized in that it contains further chemotherapeutic agents.

16. A pharmaceutical composition according to any one of claims 13 to 15 in the form of tablets, dragees, capsules, dripping solutions, suppositories, injection or infusion preparations for peroral, rectal or parenteral use.

17. Use of a compound according to any one of claims 1 to 12 together with suitable additives or excipients for the manufacture of a medicament for the treatment of tumor diseases.

18. Use of a compound according to any one of claims 1 to 12 together with suitable additives or excipients for the manufacture of a medicament for the treatment of leukemia.

19. Use according to claim 17 or 18, wherein the compound is in the form of a depot substance or as a precursor, together with a suitable, pharmaceutically acceptable diluting solution or vehicle.