Title of Invention	BETULINC ACID DERVATIVE
Abstract	BETULINIC ACID DERIVATIVE The present invention relates to novel betulinic acid derivatives with increased activity for the treatment of carcinomas and HIV diseases, a method for producing such novel betulinic acid derivatives as well as their use as pharmaceuticals.

Full Text

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to novel betulinic acid derivatives with increased activity for the treatment of carcinomas and HIV diseases, a method for preparing such novel betulinic acid derivatives as well as their use as Pharmaceuticals. Like betulinic acid, the compounds according to the invention are suitable for clinical use in men and animals to inhibit the growth of various tumors (melanomas, sarcomas, lymphomas, squam-ous carcinomas and other tumors mentioned below) as well as for the treatment of HIV diseases and - due to their antiphlogistic activity - unspecific inflammatory diseases. SUMMARY OF THE INVENTION The invention relates to novel betulic acid derivatives having the general formula (I) (Figure Removed) wherein Rx represents a hydroxy group, an amino group, a protected hydroxy group or a protected amino group. Suitable protective groups are known from the prior art, e.g., from Chapters 2 and 7 of "Protective Groups in Organic Synthesis", T.W. Greene and P.G.M. Wuts, 3Ld Edition, John Wiley & Sons, Inc. (1999), that disclosure, thus, being incorporated in the present description by reference; and R2 (Figure Removed) In particular, the present invention relates to novel betulic acid derivatives having the general formula (I) as indicated above, wherein RI represents a hydroxy group, an amino group or one of the following protected hydroxy or amino groups: (Figure Removed) and R2 is as defined above. The compounds according to the invention are novel betulic acid derivatives with increased activity and better solubility in polar solvents, and hence markedly improved application options. It goes without saying that any possible salts and inclusion compounds of the compound according to the invention are covered by the aforementioned definition of general formula (I). The present invention further relates to a method for preparing compounds of the general formula (I), wherein a betulic acid halide, particularly a betulic acid chloride, appropriately protected by the substituent RI is reacted with an alcohol or amine appropriately substituted to provide the substituent R2. If desired, the thus obtained compound of the general formula (I) wherein Rj represents a protected hydroxy or amino group may then be deprotected, by measures known from the prior art, as a function of the selected protective group to provide a compound of the general formula (I) wherein RI represents hydroxy or amino. The natural substance betulic acid, which serves as the starting and comparative compound for the compounds according to the invention, is a triterpene that was isolated already at the beginning of the last century. The name is derived from the respective alcohol betulin, an ingredient of birch bark, where it occurs in large quantities. Betulic acid exhibits anti-malaria, anti-inflammatory, anti-HIV and anti-tumor effects, which have been described in a great number of different publications. Used as a chemotherapeutic, betulic acid induces the so-called programmed cell death, also referred to as apoptosis, in tumor cells of various origins (e.g., melanoma cells). In melanomas, antiproliferative effects were detected in human mouse models both in vitro and in vivo. Particularly in melanoma cells, betulic acid appears to specifically induce an apoptotic cell destruction in tumor cells, while melanocytes are largely resistant to this substance. To date, hardly any data on possible .•;ynergd stic effects between betulic acid and other cytostatics have been available. The molecular action mechanisms in infantile tumors (Ewing sarcoma, medulloblastoma) as well as in glio-blastomas were particularly well investigated by the research group around S. Fu.lda (children's hospital, Ulm) . Other known effects of betulic acid and its known derivatives, which have been well documented in Lhe scientific literature, include its activity against HIV viruses, whose replication and receptor binding they are able to suppress, as well as their anti-inflammatory activity, which is, for instance, described by way of a mouse-ear inflammation model. Due to its antiprolifer-ative action against various tumors (melanomas, neuroectodermal tumors, sarcomas), which has been described both in animal experiments and in cell cultures, betulic acid is, thus, an extremely interesting substance for both single and possible combination therapies with other cytostatically active substances and cell-death-modulating substances, e.g., antisensoli-gonucleotides against various anti-apoptotic Bcl-2 family members and, in particular, Bcl-2, Bcl-xL as well as Mcl-1. Publications on the action mechanism of betulic acid primarily in melanomas as well as with Ewing sarcoma, glioblastoma and medulloblastoma have demonstrated that its effects are to a substantial extent caused by the induction of apoptosis on a mito-chondrial level. It is still unclear today what primary points of attack it has within the cell; amongst others neither any binding sites (receptors), if present, nor the initial signal pathways have been sufficiently explored. However, the inventors and other authors were able to demonstrate that betulic acid induces apoptosis in malign cells, yet human melanocytes and even normal cells appear to be less sensitive than malign cells. That observation is of interest, above all, also because animal-experimental data in mice have not revealed any remarkable tox-icity. Apart from those data acquired in cell cultures there are clues to the effect that betulic acid is more strongly concentrated in tumor tissues than in normal tissues. The induction of various Bcl-2 family members both in melanoma cells and in normal melanocytes and in sarcoma lines were investigated too. It was found that the expression of the anti-apoptotic protein Mcl-1. can be induced within a few hours by betulic acid. The other examined proteins of this gene family, above all Bcl-2 and Bcl-x, as well as the expression of the p53 protein remained unchanged under that treatment. The data from the scientific literature have indicated that the effects of betulic acid do not depend on the p53 protein. Since it has been recently shown that Bcl-2 and Bcl-xL are able to inhibit betulic-acid-induced apop-tosis, these observations suggest a combination of betulic acid with Bcl-2 and/or Bcl-xL antagonization, e.g., by antisensoligo-nucleotide (ASO). The same considerations apply to possible combinations with, e.g., Mcl-1 ASOs. A property of betulic acid of potential clinical relevance is the recent observation that its cytotoxicity is enhanced in low-pH culture medium. The pH is in many tumors lower than in normal tissues (Noda Y. et al, 1997). The same authors found that betulic acid is more active against resting cells than against cells in their growth phases. This property might be of additional relevance in clinical use, since many chemotherapeutics as well as radiotherapy are less effective against resting and/or acidotic cell populations. At present, melanomas, neuroectodermal tumors as well as sarcomas and HIV have above all been the most intensively investigated. These are tumors which are particularly difficult to treat and of which primarily the generalized disease forms offer hardly promising treatment options. In patients suffering from metastasized melanomas, therapeutical options are largely limited to a few substances. These include 5-(3,3-dimethyl-2-triaze-nyl)-l-H-imidazole-4-carboxamide (Dacarbazine, DTIC). Dacarba-zine still constitutes the most effective monotherapy in melanoma, exhibiting response rates in the order of about 30%. Combination therapies with other synthetic or recombinant substances, e.g., BCNU, Cisplatine, Tamoxifen, Interferon-alpha and Interleukin-2 show higher response rates in some clinical studies. These are, however, limited in time and entail elevated toxicities. Some substances derived from natural products such as, e.g., Adriamycin, Bleomycin, Etoposide and others have been investigated in respect to their activities against melanomas and their toxicities. However, in the end none of these substances turned out to be convincing in clinical daily routine. DESCRIPTION OF ILLUSTRATIVE EXAMPLES In the following, the method according to the invention will be explained in more detail by way of some examples, whereby the disclosure of the invention is not limited to these examples. 1) Acetyl betulic acid-2-amino-3-hydroxy-2-hydroxymethyl propyl ester IV (Compound B) (Figure Removed) The synthesis of acetyl betulic acid-2-amino-3-hydroxy-2-hy-droxymethyl propyl ester IV is performed by departing from betulic acid !_ vi a the intermediate stages of acetyl betulic acid II and the respective acid chloride III, by reacting the acid chloride III with trishydroxymethylaminomethane. a) Acetyl betulic acid (MW 498.74) II Two grains of betulic acid I. (MW 456.70) in 50 ml acetic anhydride are heated at reflux for 2 hours. After cooling, the reaction is poured into ice water under vigorous stirring, filtered, and the obtained solid is washed with water until the acetic acid smell has disappeared. The solid is then heated at reflux in 70% ethanol for 4 hours under stirring. After cooling, the reaction solution is filtered, the mother liquor is slightly concentrated, cooled in an ice bath and filtered once again. Yield: 86%; mp.: 290°C b) Acetyl betulic acid chloride (MW 517.18) III Two grams of acetyl betulic acid II are provided in dry benzene and supplemented with a 10-fold excess of oxalyl chloride (3.4 ml). The reaction mixture is stirred for 8 hours under cooling, and the solvent as well as excess oxalyl chloride are subsequently evaporated on a rotary evaporator. In order to remove any oxalyl chloride residues, another 20 ml of benzene are added and again evaporated under vacuum. c) Acetyl betulic acid-2-amino-3-hydroxy-2-hydroxymethyl propyl ester (MW 601.86) IV, (Compound B) The acid chloride obtained from 1 g acetyl betulic acid (.about 0.002 mol) by the method according to b) is reacted without further purification. To this end, it is dissolved in 35 ml dioxan (dry), and tris(hydroxymethyl)-aminomethane is added in double excess (0.004 mol, 0.5 g). After the addition of a spatula tip of DMAP and 3 drops of pyridine, the reaction mixture is stirred for 2 days at room temperature. After this, the solids are filtered off, the solution is concentrated on the rotary evaporator and taken up in chloroform. This chloroform solution is washed free of pyridine with 1% hydrochloric acid, water and saturated saline solution several times. After drying over Na2S04, the solvent is removed and the product is purified over a silica gel column or (and) chro- matotron. A chloroform-methanol mixture at a ratio of 10:1 was used as an eluant. Yield: 20%, mp: 156°C. 2) Acetyl betulic acid-N-(1,1-bis(hydroxymethyl)-2-hydroxy-ethyl)formamide (MW 601.86), (Compound C) (Figure Removed) The synthesis of acetyl betulinic acid N-(1,1-bis(hydroxymethyl) -2-hydroxyethyl)formamide (compound C) is carried out in a manner analogous to reaction scheme I, departing from betulic acid I. via the intermediate stages of acetyl betulic acid II and the respective acid chloride III, by reacting the acid chloride III with tris(hydroxymethyl)aminomethane. The acid chloride obtained from I g acetyl betulic acid (about 0.002 mol) according to the method of l)b) is reacted without further purification. To this end, it is dissolved in 35 ml anhydrous dioxan, and an equimolar amount of tris-(hydroxymethyl) aminomethane (0.002 mol, 0.25 g) is added. After the addition of a spatula tip of DMAP and 3 drops of pyridine, the reaction mixture is heated to 80°C for 8 hrs. After this, the reaction solution is concentrated on the rotary evaporator and the residue is taken up in chloroform. This chloroform solution is washed free of pyridine with 1% hydrochloric acid, water and saturated saline solution several times. After drying over Na2S04, the solvent is removed and the product is purified over a silica gel column or (and) chromatotron. A chloroform-methanol mixture at a ratio of 10:1 was used as an eluant. Yield: 15%, mp: 184°C. In the following, the use of the compounds according to the invention for the inhibition of tumor growths are described in comparison to betulic acid (comparative compound A). Growth inhibition in various tumor lines Examples 1 and 2 show direct comparisons between comparative compound A (betulic acid) and a novel derivative (compound B) in paired growth assays (cell count decrease after 3 days after a single application of the indicated concentrations of the respective compounds on day 1). Example 1: Comparison of compound (A) (betulic acid) with compound (B) (protected betulic acid trisester) - Growth inhibition in melanoma cell line 518A2 (obtained from Peter Schrier, Leiden, The Netherlands) after 48 hrs. The X-axis indicates the concentration in ug/ml of compounds A and B, respectively, the Y-axis gives the cell survival rate in percent of the untreated controls. The ED50 for comparative compound (A) is about 5 pg/ml, that for compound (B) about 0.7 ug/ml. (Figure Removed) Compound (B) : Compound (B), thus, exhibits a clearly higher activity than comparative compound (A), i.e., up to 7 times higher. A similarly enhanced activity is also observed with sarcomas, as shown below by way of examples on a cell line. Example 2: Comparison of the efficacy of comparative compound (A) (betulic acid) with that of compound (B) (protected betulic acid trisester) in a liposarcoma line (ATCC HTB-92). The X-axis indicates the concentration in ug/ml of compounds A and B, respectively, the Y-axis gives the cell survival rate in percent of the untreated controlThe ED50 for comparative compound (A) is about 2.5 pg/ml, that for compound (B) is about 0.9 ug/ml. Example 3: Comparison of the effects of different betulic acid derivatives according to the invention and of the comparative compound (betulic acid), respectively, on various melanoma-sarcoma lines. The data each refer to cell survival rates in percent (mean values) as against untreated cell cultures. 3a) Comparative compound (A) (betulic acid) - Effect on liposar-coma line ATCC HTB-92 Cone, yg/ml survival in % 0.3 98.0 0,6 92.8 1 92.3 1.25 79.5 2.5 49.3 5 20.8 10 15.7 survival in % 89.5 70.7 12.0 2.9 3.0 0.0 0.0 3b) Compound (B) (protected betulic acid trisester) - Effect on liposarcoma line ATCC HTB-92 Cone, ug/ml 0.3 0.6 1 1.25 2.5 5 10 3c) Compound (B) (protected betulic acid trisester) - Effect on melanoma line 518A2 Cone, ug/ml survival in % 0.3 83.0 0.6 56.3 1 11.3 1.25 10.3 2.5 2.8 5 0.0 10 0.0 3d) Compound (C) (protected betulic acid trisester) - Effect on melanoma line 518A2 and liposarcoma line ATCC HTB-92 Compound (C): (Figure Removed) Cone, ug/ml 0.25 0.5 I survival in 96 93 67.5 % melanoma Cone, pg/ml 0.3 0.6 1.0 survival in 96 82 81 % liposarcoma Beispiel 4: Preparation of a inclusion compound comprising compound (B) (protected betulinic acid trisester) in HBC (2-hydrox-propyl-beta-cyclodextrin, Sigma, Cat. No. H-107) or in HGC (2-hydroxypropyl-gamma-cyclodextrin, Sigma, Cat. No. H-125). Compound (B): (Figure Removed) H2 The respective stock solutions for further dilution are indicated below. Fifty or 100 mg/ml of compound (B) were each dissolved in EtOH. HBC was separately dissolved in water or in water plus EtOH. The further mode of procedure was basically the same as described in the data sheet supplied by Sigma. No attempts were made to adjust the pH. The solubility of compound (B) in aqueous solutions by means of inclusion in HBC or similar compounds definitely remains to be further improved, e.g., in gamma-cyclodextrin. Test #l: compound (B) 50 mg/ml in EtOH dissolved as usual Test #2: compound (B) 5.9 mg/ml in 266 mg/ml HBC in H20 Test #3: compound (B) 20 mg/ml in 266 mg/ml HBC in 50% EtOH / 50% H20 Test #4: compound (B) 25 mg/ml in 200 mg/ml HGC in 50% EtOH / 50% H20 The stock solutions indicated above were diluted to 1 mg/ml in EtOH and subsequently directly added to the culture medium. 4a) Compound (B) (protected betulinic acid trisester) as inclusion compound in HBC Survival in percent as with all other data after 3 days of cell culture after a single application on day 1 Test #1 Cone, ug/ml Survival in % melanoma 1.0 50 2.0 0.5 3.0 0.0 4.0 0.0 Test #2 Cone, ug/ml Survival in % melanoma 1.0 65 2.0 25 3.0 0.0 4.0 0.0 Test #3 Cone, ug/ml Survival in % melanoma 1.0 77 2.0 24 3.0 0.0 4.0 0.0 Test #4 Conc, pg/ml Survival in % melanoma 1.0 59 2.0 4 3.0 0.0 4b) Tolerance of compound (B) (protected betulinic acid tris-ester) as inclusion compound in H6C (2-hydroxypropyl-gamma-cyc-lodextrin) in a mouse model: Methodology: A HGC/compound (B) complex dissolved in water was intravenously injected into the tail vein of pathogen-free female C.B.-17 scid/scid (SCID) mice (4-6 weeks old, Harlan Winkelmann, Borchen, Germany). The inclusion compound was prepared as described in Example 4. The alcohol portion was removed by freezedrying of the mixture and subsequent reconstitution in water. The total volume per injection for the experiments indicated below was 200 pi. Test # 1 Initially, two mice were each treated with 100 pg HGC/compound (B) complex three times in a row on every third day. In case of good tolerance, the next pair of mice was treated with the next higher dose as indicated in the scheme below. Scheme: Group A: 3 times 100 pg HGC/compound (B) complex Group B 3 times 200 pg HGC/compound (B) complex Group C 3 times 400 pg HGC/compound (B) complex, then continuation with group A treated first: Group A 3 times 800 pg HGC/compound (B) complex Group B 3 times 1500 pg HGC/compound (B) complex; then discontinuation because of local irritation phenomena. The tests were discontinued at 1500 pg HGC/compound (B) complex because of strong local irritation phenomena in one of two mice, yet without any apparent systemic toxicity. Overall, an amount of 800 ug HGC/compound (B) complex could be administered per injection per mouse without apparent toxicity. At higher concentrations (about >1.5 mg HGC/compound (B) complex per mouse) , local irritation phenomena were observed. It is conceivable that slower infusion rates may help avoid such side effects. Test #2 In a test group of 3 mice, 800 pg HGC/compound (B) complex (in 200 pi volume) was each administered per mouse every third day, six times in a row. Also this showed no apparent clinically toxic effects. The subsequent autopsy did not suggest any macroscopic organ changes. We claim: 1. A betulinic acid compound having a formula (I): (Formula Removed) wherein R1 is a hydroxy group, an amino group, a protected hydroxy group, a protected amino group; and (Formula Removed) R2 is one of: 2. The compound as claimed in claim 1, wherein R1 is one the following: (Formula Removed) 3. A method for preparing a compound as claimed in claim 1 comprising reacting a betulic acid halide protected by a substituent R1, the substiuent Ri further defined as a protected hydroxyl or amino group, with an alcohol or amine appropriately substituted to provide the substituent R2. 4. The method as claimed in claim 3, comprising deprotecting the protected hydroxy or amino group at R1 to provide a compound of formula (I), wherein R1 is a hydroxy or amin group. 5. The compound as claimed claims 1 and 2 whenever used for the purposes of the preparation of a medicament for inhibiting the growth of tumors like melanomas, neuroectodermal tumors, sarcomas and/or effective against HIV diseases in mammals.

Documents:

3412-delnp-2005 -abstract.pdf

3412-DELNP-2005-Abstract-(23-12-2008).pdf

3412-DELNP-2005-Claims-(23-12-2008).pdf

3412-DELNP-2005-Claims-(26-12-2008).pdf

3412-delnp-2005-claims.pdf

3412-delnp-2005-correspondece-others.pdf

3412-DELNP-2005-Correspondence-Others-(01-02-2008).pdf

3412-DELNP-2005-Correspondence-Others-(01-12-2005).pdf

3412-DELNP-2005-Correspondence-Others-(23-12-2008).pdf

3412-DELNP-2005-Correspondence-Others-(24-12-2008).pdf

3412-DELNP-2005-Correspondence-Others-(26-12-2008).pdf

3412-DELNP-2005-Correspondence-Others-(28-01-2008).pdf

3412-delnp-2005-description (complete).pdf

3412-DELNP-2005-Drawings-(23-12-2008).pdf

3412-DELNP-2005-Form-1-(23-12-2008).pdf

3412-delnp-2005-form-1.pdf

3412-delnp-2005-form-18.pdf

3412-DELNP-2005-Form-2-(23-12-2008).pdf

3412-delnp-2005-form-2.pdf

3412-DELNP-2005-Form-3-(01-02-2008).pdf

3412-DELNP-2005-Form-3-(01-12-2008).pdf

3412-DELNP-2005-Form-3-(23-12-2008).pdf

3412-delnp-2005-form-3.pdf

3412-delnp-2005-form-5.pdf

3412-DELNP-2005-GPA-(26-12-2008).pdf

3412-delnp-2005-gpa.pdf

3412-delnp-2005-pct-210.pdf

3412-delnp-2005-pct-304.pdf

3412-delnp-2005-pct-306.pdf

3412-delnp-2005-pct-338.pdf

3412-delnp-2005-pct-409.pdf

3412-DELNP-2005-Petition-137-(24-12-2008).pdf